Novel methods of protein evolution

ABSTRACT

The present invention is relevant to proteins and novel methods of protein evolution. The present invention further relates to methods of identifying and mapping mutant polypeptides formed from, or based upon, a template polypeptide.

FIELD OF THE INVENTION

In a particular aspect, the present invention is relevant to proteinsand to their generation by protein evolution.

BACKGROUND

Protein engineering via site-directed mutagenesis and, more recently,molecular evolution has been successfully employed to improve enzymaticproperties in industrial applications and therapeutic properties inantibodies. Characteristics such as thermostability, pH optimum,enantioselectivity, specificity and binding affinity have all beenaltered to better adapt proteins and antibodies for specific purposes.

Since its inception, many different methods for molecular evolution havebeen described and applied to improve characteristics of the targetprotein. For example, sets of single point mutants can be generated andscreened for upmutants. Beneficial single amino acid substitutions canthen be recombined and screened to further optimize the desiredcharacteristics in the target molecule.

However, the successful evolution of a target molecule starting withsingle point mutations requires that the (sometimes) subtle changes inperformance can be accurately measured to identify the upmutants. Incases where a sensitive assay does not exist, single point mutationscannot be successfully screened. Simultaneous mutations of several sitescan be done, however the number of combinations created, increases veryquickly and reaches the limits of cloning efficiency and screeningcapability.

SUMMARY OF THE INVENTION

The present invention relates to comprehensive methods of identifyingand mapping mutant polypeptides formed from, or based upon, a templatepolypeptide. Typically, the polypeptide will comprise n amino acidresidues, wherein the method comprises (a) generating n separate sets ofpolypeptides, each set comprising member polypeptides having X number ofdifferent predetermined amino acid residues at a single predeterminedposition of the polypeptide; wherein each set of polypeptides differs inthe single predetermined position as confirmed by sequencing or othertechnique; assaying each set for at least one and preferably twopredetermined properties, characteristics or activities; (b) for eachmember identifying any change in said property, characteristic oractivity relative to the template polypeptide; and (c) creating afunctional map reflecting such changes. The number of different memberpolypeptides generated is equivalent to n×X

In the alternative, the method comprises generating a single populationcomprising the sets of mutated polypeptides. In this embodiment, theentire population is sequenced, tested for expression and screened for afunction, the individual members identified, and, preferably, thefunctional map generated.

Typically, where each naturally occurring amino acid is used, X will be19 (representing the 20 naturally occurring amino acid residues andexcluding the particular residue present in a given position of thetemplate polypeptide). However, any subset of amino acids may be usedthroughout, and each set of polypeptides may be substituted with all ora subset of the total X used for the entire population.

Any mutational or synthetic means may be used to generate the set ofmutants. In one embodiment, the generation of polypeptides comprises (i)subjecting a codon-containing polynucleotide encoding for the templatepolypeptide to polymerase-based amplification using a 63-fold degenerateoligonucleotide for each codon to be mutagenized, wherein each of the63-fold degenerate oligonucleotides is comprised of a first homologoussequence and a degenerate N,N,N triplet sequence, so as to generate aset of progeny polynucleotides; and (ii) subjecting the set of progenypolynucleotides to clonal amplification such that polypeptides encodedby the progeny polynucleotides are cloned, sequenced, expressed andscreened.

In one embodiment, the entire polypeptide is subjected to comprehensivemutagenesis. In another embodiment, one or more regions are selected forcomprehensive mutagenesis. In such case, n represents a subset or regionof the template polypeptide. For example, where the polypeptide is anantibody, the entire antibody or one or more complementarity determiningregions (CDRs) of the antibody are subjected to comprehensivemutagenesis.

The invention thus includes methods of mapping a set of mutantantibodies formed from a template antibody having at least one, andpreferably six, complementarity determining regions (CDRs), the CDRstogether comprising n amino acid residues, the method comprising (a)generating n separate sets of antibodies, each set comprising memberantibodies having X number of different predetermined amino acidresidues at a single predetermined position of the CDR; wherein each setof antibodies differs in the single predetermined position; and thenumber of different member antibodies generated is equivalent to n×X;(b) confirming by sequencing or other method that each member antibodyhas been made; (c) expressing each member antibody; (d) assaying eachset for at least one predetermined property, characteristic or activity;(e) for each member identifying any change in a property, characteristicor activity relative to the template polypeptide; and (f) creating astructural positional map of such changes. For antibodies, thepredetermined property, characteristic or property may be bindingaffinity and/or immunogenicity. As set forth above, in the alternative asingle population comprising all sets of mutated antibodies may begenerated.

In addition, provided are methods of producing a set of mutantantibodies formed from a template antibody having at least onecomplementarity determining region (CDR), the CDR comprising n aminoacid residues, the method comprising: (a) generating n separate sets ofantibodies, each set comprising member antibodies having X number ofdifferent predetermined amino acid residues at a single predeterminedposition of the CDR; wherein each set of antibodies differs in thesingle predetermined position; and the number of different memberantibodies generated is equivalent to n×X. In another embodiment,antibody comprises six CDRs, and together the CDRs comprise n amino acidresidues.

One embodiment of the disclosure includes a functional positional map(EvoMap™) made by the methods described herein.

In an additional embodiment, certain residues particularly sensitive tochange may be so indicated on the EvoMap™. Further optimization may beimplemented by making additional mutational changes at positions outsideof these sensitive positions.

In a specific embodiment, the mutations generated in the comprehensiveevolution techniques of the disclosure are confirmed by sequencing, orsome other method.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates how comprehensive positional evolution (CPE™) is usedto generate a molecule specific database (EvoMap™).

FIG. 2 shows an example of a EvoMap™ and how additional optimization maybe implemented by Synergy evolution.

FIG. 3 shows the expression levels of full length IgGs derived from anFc codon variant library compared to the expression level of thewild-type IgG in the same mammalian cell line.

FIG. 4 shows a schematic of Comprehensive Positional Insertion (CPI™)evolution.

FIG. 5 illustrates one combination of evolution methods: a lengthenednucleic acid from CPI™ evolution is subjected to ComprehensivePositional Evolution (CPE™) and used to generate a molecule specificdatabase (EvoMap™)

FIG. 6 shows a schematic of Comprehensive Positional Deletion (CPD™)evolution.

FIG. 7 illustrates another combination of evolution methods: a shortenednucleic acid from CPD™ evolution is subjected to ComprehensivePositional Evolution (CPE™) and used to generate a molecule specificdatabase (EvoMap™).

FIG. 8 shows a schematic of Comprehensive Positional Synthesis (CPS™)which can be used to combine upmutants from CPE™

FIG. 9 shows a schematic of a hypothetical three-dimensional EvoMap™.

DEFINITION OF TERMS

In order to facilitate understanding of the examples provided herein,certain frequently occurring methods and/or terms will be described.

The term “agent” is used herein to denote a polypeptide, a mixture ofpolypeptides, an array of spatially localized compounds (e.g., a VLSIPSpeptide array, polynucleotide array, and/or combinatorial small moleculearray), biological macromolecule, a bacteriophage peptide displaylibrary, a bacteriophage antibody (e.g., scFv) display library, apolysome peptide display library, or an extract made form biologicalmaterials such as bacteria, plants, fungi, or animal (particularmammalian) cells or tissues. Agents are evaluated for potential activityas anti-neoplastics, anti-inflamnmatories or apoptosis modulators byinclusion in screening assays described hereinbelow. Agents areevaluated for potential activity as specific protein interactioninhibitors (i.e., an agent which selectively inhibits a bindinginteraction between two predetermined polypeptides but which doe snotsubstantially interfere with cell viability) by inclusion in screeningassays described hereinbelow.

The term “amino acid” as used herein refers to any organic compound thatcontains an amino group (—NH₂) and a carboxyl group (—COOH); preferablyeither as free groups or alternatively after condensation as part ofpeptide bonds. The “twenty naturally encoded polypeptide-formingalpha-amino acids” are understood in the art and refer to: alanine (alaor A), arginine (arg or R), asparagine (asn or N), aspartic acid (asp orD), cysteine (cys or C), gluatamic acid (glu or E), glutamine (gln orQ), glycine (gly or G), histidine (his or H), isoleucine (ile or I),leucine (leu or L), lysine (lys or K), methionine (met or M),phenylalanine (phe or F), proline (pro or P), serine (ser or S),threonine (thr or T), tryptophan (trp or W), tyrosine (tyr or Y), andvaline (val or V).

The term “amplification” means that the number of copies of apolynucleotide is increased.

The term “antibody”, as used herein, refers to intact immunoglobulinmolecules, as well as fragments of immunoglobulin molecules, such asFab, Fab′, (Fab′)₂, Fv, and SCA fragments, that are capable of bindingto an epitope of an antigen. These antibody fragments, which retain someability to selectively bind to an antigen (e.g., a polypeptide antigen)of the antibody from which they are derived, can be made using wellknown methods in the art (see, e.g., Harlow and Lane, supra), and aredescribed further, as follows. Antibodies can be used to isolatepreparative quantities of the antigen by immunoaffinity chromatography.Various other uses of such antibodies are to diagnose and/or stagedisease (e.g., neoplasia) and for therapeutic application to treatdisease, such as for example: neoplasia, autoimmune disease, AIDS,cardiovascular disease, infections, and the like. Chimeric, human-like,humanized or fully human antibodies are particularly useful foradministration to human patients.

An Fab fragment consists of a monovalent antigen-binding fragment of anantibody molecule, and can be produced by digestion of a whole antibodymolecule with the enzyme papain, to yield a fragment consisting of anintact light chain and a portion of a heavy chain.

An Fab′ fragment of an antibody molecule can be obtained by treating awhole antibody molecule with pepsin, followed by reduction, to yield amolecule consisting of an intact light chain and a portion of a heavychain. Two Fab′ fragments are obtained per antibody molecule treated inthis manner.

An (Fab′)₂ fragment of an antibody can be obtained by treating a wholeantibody molecule with the enzyme pepsin, without subsequent reduction.A (Fab′)₂ fragment is a dimer of two Fab′ fragments, held together bytwo disulfide bonds.

An Fv fragment is defined as a genetically engineered fragmentcontaining the variable region of a light chain and the variable regionof a heavy chain expressed as two chains.

A single chain antibody (“SCA”) is a genetically engineered single chainmolecule containing the variable region of a light chain and thevariable region of a heavy chain, linked by a suitable, flexiblepolypeptide liner.

The term “biosimilar”, also termed “follow-on biologic”, refers toofficially approved new versions of innovator biopharmaceuticalproducts, following patent or exclusivity expiry.

The term “cell production host”, or “manufacturing host”, refers to acell line used for the production or manufacturing of proteins.Eukaryotic cells such as mammalian cells, including, but not limited tohuman, mouse, hamster, rat, monkey cell lines as well as yeast, insectand plant cell lines. Prokaryotic cells can alternatively be utilized.In one aspect, a mammalian cell production host is selected from amember of the group consisting of 3T3 mouse fibroblast cells; BHK21Syrian hamster fibroblast cells; MDCK, dog epithelial cells; Hela humanepithelial cells; PtK1 rat kangaroo epithelial cells; SP2/0 mouse plasmacells; and NS0 mouse mouse plasma cells; HEK 293 human embryonic kidneycells; COS monkey kidney cells; CHO, CHO-S Chinese hamster ovary cells;R1 mouse embryonic cells; E14.1 mouse embryonic cells; H1 humanembryonic cells; H9 human embryonic cells; PER C.6, human embryoniccells. In another aspect, the cell production host is a GS-NS0 orGS-CHOK1 cell line. In another aspect, the cell production host isselected from S. cerevisiae yeast cells; and picchia yeast cells. Inanother aspect, the cell production host is a bacterial cell line.

A molecule that has a “chimeric property” is a molecule that is: 1) inpart homologous and in part heterologous to a first reference molecule;while 2) at the same time being in part homologous and in partheterologous to a second reference molecule; without 3) precluding thepossibility of being at the same time in part homologous and in partheterologous to still one or more additional reference molecules. In anon-limiting embodiment, a chimeric molecule may be prepared byassemblying a reassortment of partial molecular sequences. In anon-limiting aspect, a chimeric polynucleotide molecule may be preparedby synthesizing the chimeric polynucleotide using plurality of moleculartemplates, such that the resultant chimeric polynucleotide hasproperties of a plurality of templates.

The term “cognate” as used herein refers to a gene sequence that isevolutionarily and functionally related between species. For example,but not limitation, in the human genome the human CD4 gene is thecognate gene to the mouse 3d4 gene, since the sequences and structuresof these two genes indicate that they are highly homologous and bothgenes encode a protein which functions in signaling T cell activationthrough MHC class II-restricted antigen recognition.

The term “commercial scale” means production of a protein or antibody ata scale appropriate for resale.

A “comparison window,” as used herein, refers to a conceptual segment ofat least 20 contiguous nucleotide positions wherein a polynucleotidesequence may be compared to a reference sequence of at least 20contiguous nucleotides and wherein the portion of the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Optimal alignment of sequences for aligning acomparison window may be conducted by the local homology algorithm ofSmith and Waterman (1981) Adv. Appl. Math. 2: 482 by the homologyalignment algorithm of Needlemen and Wuncsch J. Mol. Biol. 48: 443(1970), by the search of similarity method of Pearson and Lipman Proc.Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package Release 7.0, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by inspection, and the bestaligmnent (i.e., resulting in the highest percentage of homology overthe comparison window) generated by the various methods is selected.

As used herein, the term “complementarity-determining region” and “CDR”refer to the art-recognized term as exemplified by the Kabat andChothia. CDR definitions are also generally known as supervariableregions or hypervariable loops (Chothia and Leks, 1987; Clothia et al.,1989; Kabat et al., 1987; and Tramontano et al., 1990). Variable regiondomains typically comprise the amino-terminal approximately 105-115amino acids of a naturally-occurring immunoglobulin chain (e.g., aminoacids 1-110), although variable domains somewhat shorter or longer arealso suitable for forming single-chain antibodies. The CDRs are parts ofimmunoglobulins that determine the specificity of said molecules andmake contact with a specific ligand. The CDRs are the most variable partof the molecule and contribute to the diversity of these molecules.There are three CDR regions CDR1, CDR2 and CDR3 in each V domain. CDR-Hdepicts a CDR region of a variable heavy chain and CDR-L relates to aCDR region of a variable light chain. H means the variable heavy chainand L means the variable light chain. The CDR regions of an Ig-derivedregion may be determined as described in Kabat (1991). Sequences ofProteins of Immunological Interest, 5th edit., NIH Publication no.91-3242 U.S. Department of Health and Human Services, Chothia (1987) J.Mol. Biol. 196, 901-917 and Chothia (1989) Nature, 342, 877-883.

The term “comprehensive” is used herein to refer to a technique ofevolution wherein every possible change is made at each position of atemplate polynucleotide or template polypeptide and the polynucleotideor polypeptide is tested to confirm the intended change has been made bysequencing or some other technique. Comprehensive mutagenesis refers tomutating the DNA of a region of a gene encoding a protein that changescodon amino acid sequence of the protein and then determining viasequencing or other technologies that all mutations have been made andin the optimal case arrayed where every clone is in an identifiableposition and/or uniquely tagged. Then screening of all of the expressedmutants is performed to ensure that all are expressed comprehensivelyfor an improved phenotype in order to provide guaranteed comprehensivecoverage, i.e. CPE library with Comprehensive Screening comprising theBioAtla CPE process. Non-expressing clones in the screening system willalso be simultaneously measured for expression to ensure that are notincorrectly labeled as negative or neutral mutations once enabled forexpression an alternative system such as in vitro transcription andtranslation. Alternatively, sequencing could be performed on all clonesafter screening, but it should include all negative, neutral andup-mutant clones. Any mutants not identified are then be added in asecond round of screening to yield and a true comprehensive mutagenesisand screening expression/activity system such as CPE. This is enabled inpart by recent successes in high throughput sequencing that did notexist previously.

“Conservative amino acid substitutions” refer to the interchangeabilityof residues having similar side chains. For example, a group of aminoacids having aliphatic side chains is glycine, alanine, valine, leucine,and isoleucine; a group of amino acids having aliphatic-hydroxyl sidechains is serine and threonine; a group of amino acids havingamide-containing side chains is asparagine and glutamine; a group ofamino acids having aromatic side chains is phenylalanine, tyrosine, andtryptophan; a group of amino acids having basic side chains is lysine,arginine, and histidine; and a group of amino acids havingsulfur-containing side chains is cysteine and methionine. Preferredconservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine.

The term “corresponds to” is used herein to mean that a polynucleotidesequence is homologous (i.e., is identical, not strictly evolutionarilyrelated) to all or a portion of a reference polynucleotide sequence, orthat a polypeptide sequence is identical to a reference polypeptidesequence. In contradistinction, the term “complementary to” is usedherein to mean that the complementary sequence is homologous to all or aportion of a reference polynucleotide sequence. For illustration, thenucleotide sequence “TATAC” corresponds to a reference “TATAC” and iscomplementary to a reference sequence “GTATA.”

The term “degrading effective” amount refers to the amount of which isrequired to process at least 50% of the substrate, as compared tosubstrate not contacted with the enzyme. Preferably, at least 80% of thesubstrate is degraded.

As used herein, the term “defined sequence framework” refers to a set ofdefined sequences that are selected on a non-random basis, generally onthe basis of experimental data or structural data; for example, adefined sequence framework may comprise a set of amino acid sequencesthat are predicted to form a β-sheet structure or may comprise a leucinezipper heptad repeat motif, a zinc-finger domain, among othervariations. A “defined sequence kernal” is a set of sequences whichencompass a limited scope of variability. Whereas (1) a completelyrandom 10-mer sequence of the 20 conventional amino acids can be any of(20)10 sequences, and (2) a pseudorandom 10-mer sequence of the 20conventional amino acids can be any of (20)10 sequences but will exhibita bias for certain residues at certain positions and/or overall, (3) adefined sequence kernal is a subset of sequences if each residueposition was allowed to be any of the allowable 20 conventional aminoacids (and/or allowable unconventional amino/imino acids). A definedsequence kernal generally comprises variant and invariant residuepositions and/or comprises variant residue positions which can comprisea residue selected from a defined subset of amino acid residues), andthe like, either segmentally or over the entire length of the individualselected library member sequence. Defined sequence kernels can refer toeither amino acid sequences or polynucleotide sequences. Of illustrationand not limitation, the sequences (NNK)10 and (NNM)10, wherein Nrepresents A, T, G, or C; K represents G or T; and M represents A or C,are defined sequence kernels.

The term “deimmunization” as used herein relates to production of avariant of the template binding molecule, which is modified compared toan original wild type molecule by rendering said variant non-immunogenicor less immunogenic in humans. Deimmunized molecules according to theinvention relate to antibodies or parts thereof (like frameworks and/orCDRs) of non-human origin. Corresponding examples are antibodies orfragments thereof as described in U.S. Pat. No. 4,361,549. The term“deimmunized” also relates to molecules, which show reduced propensityto generate T cell epitopes. In accordance with this invention, the term“reduced propensity to generate T cell epitopes” relates to the removalof T-cell epitopes leading to specific T-cell activation.

Furthermore, reduced propensity to generate T cell epitopes meanssubstitution of amino acids contributing to the formation of T cellepitopes, i.e. substitution of amino acids, which are essential forformation of a T cell epitope. In other words, reduced propensity togenerate T cell epitopes relates to reduced immunogenicity or reducedcapacity to induce antigen independent T cell proliferation. Inaddition, reduced propensity to generate T cell epitopes relates todeimmunization, which means loss or reduction of potential T cellepitopes of amino acid sequences inducing antigen independent T cellproliferation.

The term “T cell epitope” as used herein relates to short peptidesequences which can be released during the degradation of peptides,polypeptide or proteins within cells and subsequently be presented bymolecules of the major histocompatibility complex (MHC) in order totrigger the activation of T cells; see inter alia WO 02/066514. Forpeptides presented by MHC class II such activation of T cells can theninduce an antibody response by direct stimulation of B cells to producesaid antibodies.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 ug of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a gel to isolate the desired fragment.

The term “DNA shuffling” is used herein to indicate recombinationbetween substantially homologous but non-identical sequences, in someembodiments DNA shuffling may involve crossover via non-homologousrecombination, such as via cer/10× and/or flp/frt systems and the like.Shuffling may be random or non-random.

As used in this invention, the term “epitope” refers to an antigenicdeterminant on an antigen, such as a phytase polypeptide, to which theparatope of an antibody, such as a phytase-specific antibody, binds.Antigenic determinants usually consist of chemically active surfacegroupings of molecules, such as amino acids or sugar side chains, andcan have specific three-dimensional structural characteristics, as wellas specific charge characteristics. As used herein “epitope” refers tothat portion of an antigen or other macromolecule capable of forming abinding interaction that interacts with the variable region binding bodyof an antibody. Typically, such binding interaction is manifested as anintermolecular contact with one or more amino acid residues of a CDR.

The term “evolution” refers to a change in at least one property,characteristic or activity of a genetically or synthetically modifiedprotein or antibody when compared to a template protein or antibody.

The terms “fragment”, “derivative” and “analog” when referring to areference polypeptide comprise a polypeptide which retains at least onebiological function or activity that is at least essentially same asthat of the reference polypeptide. Furthermore, the terms “fragment”,“derivative” or “analog” are exemplified by a “pro-form” molecule, suchas a low activity proprotein that can be modified by cleavage to producea mature enzyme with significantly higher activity.

A method is provided herein for producing from a template polypeptide aset of progeny polypeptides in which a “full range of single amino acidsubstitutions” is represented at each amino acid position. As usedherein, “full range of single amino acid substitutions” is in referenceto the naturally encoded 20 naturally encoded polypeptide-formingalpha-amino acids, as described herein.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

“Genetic instability”, as used herein, refers to the natural tendency ofhighly repetitive sequences to be lost through a process of reductiveevents generally involving sequence simplification through the loss ofrepeated sequences. Deletions tend to involve the loss of one copy of arepeat and everything between the repeats.

The term “heterologous” means that one single-stranded nucleic acidsequence is unable to hybridize to another single-stranded nucleic acidsequence or its complement. Thus, areas of heterology means that areasof polynucleotides or polynucleotides have areas or regions within theirsequence which are unable to hybridize to another nucleic acid orpolynucleotide. Such regions or areas are for example areas ofmutations.

The term “homologous” or “homeologous” means that one single-strandednucleic acid nucleic acid sequence may hybridize to a complementarysingle-stranded nucleic acid sequence. The degree of hybridization maydepend on a number of factors including the amount of identity betweenthe sequences and the hybridization conditions such as temperature andsalt concentrations as discussed later. Preferably the region ofidentity is greater than about 5 bp, more preferably the region ofidentity is greater than 10 bp.

The term “humanized” is used to describe antibodies whereincomplementarity determining regions (CDRs) from a mammalian animal,e.g., a mouse, are combined with a human framework region. Oftenpolynucleotides encoding the isolated CDRs will be grafted intopolynucleotides encoding a suitable variable region framework (andoptionally constant regions) to form polynucleotides encoding completeantibodies (e.g., humanized or fully-human), antibody fragments, and thelike. In another aspect, besides mouse antibodies, other species can behumanized, such as, for example, other rodent, camel, rabbit, cat, dog,pig, horse, cow, fish, llama and shark. In a broad aspect, any speciesthat produces antibodies can be utilized in the production of humanizedantibodies. Additionally, the antibodies of the invention may bechimeric, human-like, humanized or fully human, in order to reduce theirpotential antigenicity, without reducing their affinity for theirtarget. Chimeric, human-like and humanized antibodies have generallybeen described in the art. By incorporating as little foreign sequenceas possible in the hybrid antibody, the antigenicity is reduced.Preparation of these hybrid antibodies may be carried out by methodswell known in the art.

An immunoglobulin light or heavy chain variable region consists of a“framework” region interrupted by three hypervariable regions, alsocalled CDR's. The extent of the framework region and CDR's have beenprecisely defined (see, “Sequences of Proteins of ImmunologicalInterest,” Kabat et al., 1987). The sequences of the framework regionsof different light or heavy chains are relatively conserved within aspecies. As used herein, a “human framework region” is a frameworkregion that is substantially identical (about 85 or more, usually 90-95or more) to the framework region of a naturally occurring humanimmunoglobulin. The framework region of an antibody, that is thecombined framework regions of the constituent light and heavy chains,serves to position and align the CDR's. The CDR's are primarilyresponsible for binding to an epitope of an antigen. In accordance withthis invention, a framework region relates to a region in the V domain(VH or VL domain) of immunoglobulins that provides a protein scaffoldfor the hypervariable complementarity determining regions (CDRs) thatmake contact with the antigen. In each V domain, there are fourframework regions designated FR1, FR2, FR3 and FR4. Framework 1encompasses the region from the N-terminus of the V domain until thebeginning of CDR1, framework 2 relates to the region between CDR1 andCDR2, framework 3 encompasses the region between CDR2 and CDR3 andframework 4 means the region from the end of CDR3 until the C-terminusof the V domain; see, inter alia, Janeway, Immunobiology, GarlandPublishing, 2001, 5th ed. Thus, the framework regions encompass all theregions outside the CDR regions in VH or VL domains.

The person skilled in the art is readily in a position to deduce from agiven sequence the framework regions and, the CDRs; see Kabat (1991)Sequences of Proteins of Immunological Interest, 5th edit., NIHPublication no. 91-3242 U.S. Department of Health and Human Services,Chothia (1987) J. Mol. Biol. 196, 901-917 and Chothia (1989) Nature,342, 877-883.

The benefits of this invention extend to “industrial applications” (orindustrial processes), which term is used to include applications incommercial industry proper (or simply industry) as well asnon-commercial industrial applications (e.g. biomedical research at anon-profit institution). Relevant applications include those in areas ofdiagnosis, medicine, agriculture, manufacturing, and academia.

The term “identical” or “identity” means that two nucleic acid sequenceshave the same sequence or a complementary sequence. Thus, “areas ofidentity” means that regions or areas of a polynucleotide or the overallpolynucleotide are identical or complementary to areas of anotherpolynucleotide or the polynucleotide.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide or proteinpresent in a living animal is not isolated, but the same polynucleotideor protein, separated from some or all of the coexisting materials inthe natural system, is isolated. Such polynucleotides could be part of avector and/or such polynucleotides or proteins could be part of acomposition, and still be isolated in that such vector or composition isnot part of its natural environment.

By “isolated nucleic acid” is meant a nucleic acid, e.g., a DNA or RNAmolecule, that is not immediately contiguous with the 5′ and 3′ flankingsequences with which it normally is immediately contiguous when presentin the naturally occurring genome of the organism from which it isderived. The term thus describes, for example, a nucleic acid that isincorporated into a vector, such as a plasmid or viral vector; a nucleicacid that is incorporated into the genome of a heterologous cell (or thegenome of a homologous cell, but at a site different from that at whichit naturally occurs); and a nucleic acid that exists as a separatemolecule, e.g., a DNA fragment produced by PCR amplification orrestriction enzyme digestion, or an RNA molecule produced by in vitrotranscription. The term also describes a recombinant nucleic acid thatforms part of a hybrid gene encoding additional polypeptide sequencesthat can be used, for example, in the production of a fusion protein.

As used herein “ligand” refers to a molecule, such as a random peptideor variable segment sequence, that is recognized by a particularreceptor. As one of skill in the art will recognize, a molecule (ormacromolecular complex) can be both a receptor and a ligand. In general,the binding partner having a smaller molecular weight is referred to asthe ligand and the binding partner having a greater molecular weight isreferred to as a receptor.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis et al., 1982, p.146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units of T4 DNA ligase (“ligase”)per 0.5 μg of approximately equimolar amounts of the DNA fragments to beligated.

As used herein, “linker” or “spacer” refers to a molecule or group ofmolecules that connects two molecules, such as a DNA binding protein anda random peptide, and serves to place the two molecules in a preferredconfiguration, e.g., so that the random peptide can bind to a receptorwith minimal steric hindrance from the DNA binding protein.

The term “mammalian cell surface display” refers to a technique wherebya protein or antibody, or a portion of an antibody, is expressed anddisplayed on a mammalian host cell surface for screening purposes; forexample, by screening for specific antigen binding by a combination ofmagnetic beads and fluorescence-activated cell sorting. In one aspect,mammalian expression vectors are used for simultaneous expression ofimmunoglobulins as both a secreted and cell surface bound form as inDuBridge et al., US 2009/0136950, which is incorporated herein byreference. In another aspect, the techniques of Gao et al. are employedfor a viral vector encoding for a library of antibodies or antibodyfragments are displayed on the cell membranes when expressed in a cellas in Gao et al., US 2007/0111260, incorporated herein by reference.Whole IgG surface display on mammalian cells is known. For example, aAkamatsuu et al. developed a mammalian cell surface display vector,suitable for directly isolating IgG molecules based on theirantigen-binding affinity and biological activity. Using an Epstein-Barrvirus-derived episomal vector, antibody libraries were displayed aswhole IgG molecules on the cell surface and screened for specificantigen binding by a combination of magnetic beads andfluorescence-activated cell sorting. Plasmids encoding antibodies withdesired binding characteristics were recovered from sorted cells andconverted to the form for production of soluble IgG. Akamatsuu et al. J.Immunol. Methods 2007 327(1-2):40-52; incorporated herein by reference.Ho et al. used human embryonic kidney 293T cells that are widely usedfor transient protein expression for cell surface display ofsingle-chain Fv antibodies for affinity maturation. Cells expressing arare mutant antibody with higher affinity were enriched 240-fold by asingle-pass cell sorting from a large excess of cells expressing WTantibody with a slightly lower affinity. Furthermore, a highly enrichedmutant was obtained with increased binding affinity for CD22 after asingle selection of a combinatory library randomizing an intrinsicantibody hotspot. Ho et al. Isolation of anti-CD22 Fv with high affinityby Fv display on human cells, Proc Natl Acad Sci USA 2006 Jun. 20;103(25): 9637-9642; incorporated herein by reference.

Beerli et al. used B cells specific for an antigen of interest whichwere directly isolated from peripheral blood mononuclear cells (PBMC) ofhuman donors. Recombinant, antigen-specific single-chain Fv (scFv)libraries are generated from this pool of B cells and screened bymammalian cell surface display by using a Sindbis virus expressionsystem. This method allows isolating antigen-specific antibodies by asingle round of FACS. The variable regions (VRs) of the heavy chains(HCs) and light chains (LCs) were isolated from positive clones andrecombinant fully human antibodies produced as whole IgG or Fabfragments. In this manner, several hypermutated high-affinity antibodiesbinding the Qβ virus like particle (VLP), a model viral antigen, as wellas antibodies specific for nicotine were isolated. All antibodies showedhigh expression levels in cell culture. The human nicotine-specific mAbswere validated preclinically in a mouse model. Beerli et al., Isolationof human monoclonal antibodies by mammalian cell display, Proc Natl AcadSci USA. 2008 Sep. 23; 105(38): 14336-14341; incorporated herein byreference.

Yeast cell surface display is also known, for example, see Kondo andUeda 2004, Yeast cell-surface display-applications of molecular display,Appl. Microbiol. Biotechnol., 64(1): 28-40, which describes for example,a cell-surface engineering system using the yeast Saccharomycescerevisiae. Several representative display systems for the expression inyeast S. cerevisiae are described in Lee et al, 2003, Microbialcell-surface display, TRENDS in Bitechnol. 21(1): 45-52. Also Boder andWittrup 1997, Yeast surface display for screening combinatorialpolypeptide libraries, Nature Biotechnol., 15(6): 553.

The term “manufacturing” refers to production of a protein at asufficient quantity to permit at least Phase I clinical testing of atherapeutic protein, or sufficient quantity for regulatory approval of adiagnostic protein.

The term “missense mutation” refers to a point mutation where a singlenucleotide is changed, resulting in a codon that codes for a differentamino acid. Mutations that change an amino acid to a stop codon arecalled nonsense mutations.

As used herein, a “molecular property to be evolved” includes referenceto molecules comprised of a polynucleotide sequence, molecules comprisedof a polypeptide sequence, and molecules comprised in part of apolynucleotide sequence and in part of a polypeptide sequence.Particularly relevant—but by no means limiting—examples of molecularproperties to be evolved include enzymatic activities at specifiedconditions, such as related to temperature; salinity; pressure; pH; andconcentration of glycerol, DMSO, detergent, and/or any other molecularspecies with which contact is made in a reaction environment. Additionalparticularly relevant—but by no means limiting examples of molecularproperties to be evolved include stabilities—e.g., the amount of aresidual molecular property that is present after a specified exposuretime to a specified environment, such as may be encountered duringstorage.

The term “Multidimensional Epitope Mapping” (MEM) refers to theidentification of the epitope and the resolution of the amino acids thatare important for antibody binding. Information about the binding sites(epitopes) of proteins recognized by antibodies is important for theiruse as biological or diagnostic tools as well as for understanding theirmechanisms of action. However, antigens are highly diverse, in theirprimary sequence as well as in three dimensional structures. Epitopesgenerally fall into 3 categories: 1) linear epitopes, i.e. the antibodybinds to residues on a linear part of the polypeptide chain, 2)conformational epitopes, where the binding site is formed by astructural element (e.g. a-helix, loop), 3) discontinuous epitopes wheretwo or more separate stretches of the polypeptide chain which arebrought together in the three dimensional structure of the antigen formthe binding surface.

The term “mutating” refers to creating a mutation in a nucleic acidsequence; in the event where the mutation occurs within the codingregion of a protein, it will lead to a codon change which may or may notlead to an amino acid change.

The term “mutations” means changes in the sequence of a wild-typenucleic acid sequence or changes in the sequence of a peptide orpolypeptides. Such mutations may be point mutations such as transitionsor transversions. The mutations may be deletions, insertions orduplications.

As used herein, the degenerate “N,N,G/T” nucleotide sequence represents32 possible triplets, where “N” can be A, C, G or T.

As used herein, the degenerate “N,N,N” nucleotide sequence represents 64possible triplets, where “N” can be A, C, G or T.

The term “naturally-occurring” as used herein as applied to the objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally occurring. Generally, the term naturally occurring refers toan object as present in a non-pathological (un-diseased) individual,such as would be typical for the species.

As used herein, a “nucleic acid molecule” is comprised of at least onebase or one base pair, depending on whether it is single-stranded ordouble-stranded, respectively. Furthermore, a nucleic acid molecule maybelong exclusively or chimerically to any group of nucleotide-containingmolecules, as exemplified by, but not limited to, the following groupsof nucleic acid molecules: RNA, DNA, genomic nucleic acids, non-genomicnucleic acids, naturally occurring and not naturally occurring nucleicacids, and synthetic nucleic acids. This includes, by way ofnon-limiting example, nucleic acids associated with any organelle, suchas the mitochondria, ribosomal RNA, and nucleic acid molecules comprisedchimerically of one or more components that are not naturally occurringalong with naturally occurring components.

Additionally, a “nucleic acid molecule” may contain in part one or morenon-nucleotide-based components as exemplified by, but not limited to,amino acids and sugars. Thus, by way of example, but not limitation, aribozyme that is in part nucleotide-based and in part protein-based isconsidered a “nucleic acid molecule”.

In addition, by way of example, but not limitation, a nucleic acidmolecule that is labeled with a detectable moiety, such as a radioactiveor alternatively a non-radioactive label, is likewise considered a“nucleic acid molecule”.

The terms “nucleic acid sequence coding for” or a “DNA coding sequenceof” or a “nucleotide sequence encoding” a particular protein—as well asother synonymous terms—refer to a DNA sequence which is transcribed andtranslated into a protein when placed under the control of appropriateregulatory sequences. A “promotor sequence” is a DNA regulatory regioncapable of binding RNA polymerase in a cell and initiating transcriptionof a downstream (3′ direction) coding sequence. The promoter is part ofthe DNA sequence. This sequence region has a start codon at its 3′terminus. The promoter sequence does include the minimum number of baseswhere elements necessary to initiate transcription at levels detectableabove background. However, after the RNA polymerase binds the sequenceand transcription is initiated at the start codon (3′ terminus with apromoter), transcription proceeds downstream in the 3′ direction. Withinthe promotor sequence will be found a transcription initiation site(conveniently defined by mapping with nuclease Si) as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

The terms “nucleic acid encoding an protein” or “DNA encoding anprotein” or “polynucleotide encoding an protein” and other synonymousterms encompasses a polynucleotide which includes only coding sequencefor the protein as well as a polynucleotide which includes additionalcoding and/or non-Cq3 coding sequence.

In one preferred embodiment, a “specific nucleic acid molecule species”is defined by its chemical structure, as exemplified by, but not limitedto, its primary sequence. In another preferred embodiment, a specific“nucleic acid molecule species” is defined by a function of the nucleicacid species or by a function of a product derived from the nucleic acidspecies. Thus, by way of non-limiting example, a “specific nucleic acidmolecule species” may be defined by one or more activities or propertiesattributable to it, including activities or properties attributable itsexpressed product.

The instant definition of “assembling a working nucleic acid sample intoa nucleic acid library” includes the process of incorporating a nucleicacid sample into a vector-based collection, such as by ligation into avector and transformation of a host. A description of relevant vectors,hosts, and other reagents as well as specific non-limiting examplesthereof are provided hereinafter. The instant definition of “assemblinga working nucleic acid sample into a nucleic acid library” also includesthe process of incorporating a nucleic acid sample into anon-vector-based collection, such as by ligation to adaptors. Preferablythe adaptors can anneal to PCR primers to facilitate amplification byPCR.

Accordingly, in a non-limiting embodiment, a “nucleic acid library” iscomprised of a vector-based collection of one or more nucleic acidmolecules. In another preferred embodiment a “nucleic acid library” iscomprised of a non-vector-based collection of nucleic acid molecules. Inyet another preferred embodiment a “nucleic acid library” is comprisedof a combined collection of nucleic acid molecules that is in partvector-based and in part non-vector-based. Preferably, the collection ofmolecules comprising a library is searchable and separable according toindividual nucleic acid molecule species.

The present invention provides a “nucleic acid construct” oralternatively a “nucleotide construct” or alternatively a “DNAconstruct”. The term “construct” is used herein to describe a molecule,such as a polynucleotide (e.g., a phytase polynucleotide) may optionallybe chemically bonded to one or more additional molecular moieties, suchas a vector, or parts of a vector. In a specific—but by no meanslimiting—aspect, a nucleotide construct is exemplified by a DNAexpression DNA expression constructs suitable for the transformation ofa host cell.

An “oligonucleotide” (or synonymously an “oligo”) refers to either asingle stranded polydeoxynucleotide or two complementarypolydeoxynucleotide strands which may be chemically synthesized. Suchsynthetic oligonucleotides may or may not have a 5′ phosphate. Thosethat do not will not ligate to another oligonucleotide without adding aphosphate with an ATP in the presence of a kinase. A syntheticoligonucleotide will ligate to a fragment that has not beendephosphorylated. To achieve polymerase-based amplification (such aswith PCR), a “32-fold degenerate oligonucleotide that is comprised of,in series, at least a first homologous sequence, a degenerate N,N,G/Tsequence, and a second homologous sequence” is mentioned. As used inthis context, “homologous” is in reference to homology between the oligoand the parental polynucleotide that is subjected to thepolymerase-based amplification.

As used herein, the term “operably linked” refers to a linkage ofpolynucleotide elements in a functional relationship. A nucleic acid is“operably linked” when it is placed into a functional relationship withanother nucleic acid sequence. For instance, a promoter or enhancer isoperably linked to a coding sequence if it affects the transcription ofthe coding sequence. Operably linked means that the DNA sequences beinglinked are typically contiguous and, where necessary to join two proteincoding regions, contiguous and in reading frame.

A coding sequence is “operably linked to” another coding sequence whenRNA polymerase will transcribe the two coding sequences into a singlemRNA, which is then translated into a single polypeptide having aminoacids derived from both coding sequences. The coding sequences need notbe contiguous to one another so long as the expressed sequences areultimately processed to produce the desired protein.

As used herein the term “physiological conditions” refers totemperature, pH, ionic strength, viscosity, and like biochemicalparameters which are compatible with a viable organism, and/or whichtypically exist intracellularly in a viable cultured yeast cell ormammalian cell. For example, the intracellular conditions in a yeastcell grown under typical laboratory culture conditions are physiologicalconditions. Suitable in vitro reaction conditions for in vitrotranscription cocktails are generally physiological conditions. Ingeneral, in vitro physiological conditions comprise 50-200 mM NaCl orKCl, pH 6.5-8.5, 20-45° C. and 0.001-10 mM divalent cation (e.g., Mg++,Ca++); preferably about 150 mM NaCl or KCl, pH 7.2-7.6, 5 mM divalentcation, and often include 0.01-1.0 percent nonspecific protein (e.g.,BSA). A non-ionic detergent (Tween, NP-40, Triton X-100) can often bepresent, usually at about 0.001 to 2%, typically 0.05-0.2% (v/v).Particular aqueous conditions may be selected by the practitioneraccording to conventional methods. For general guidance, the followingbuffered aqueous conditions may be applicable: 10-250 mM NaCl, 5-50 mMTris HCl, pH 5-8, with optional addition of divalent cation(s) and/ormetal chelators and/or non-ionic detergents and/or membrane fractionsand/or anti-foam agents and/or scintillants.

The term “population” as used herein means a collection of componentssuch as polynucleotides, portions or polynucleotides or proteins. A“mixed population: means a collection of components which belong to thesame family of nucleic acids or proteins (i.e., are related) but whichdiffer in their sequence (i.e., are not identical) and hence in theirbiological activity.

A molecule having a “pro-form” refers to a molecule that undergoes anycombination of one or more covalent and noncovalent chemicalmodifications (e.g., glycosylation, proteolytic cleavage, dimerizationor oligomerization, temperature-induced or pH-induced conformationalchange, association with a co-factor, etc.) en route to attain a moremature molecular form having a property difference (e.g. an increase inactivity) in comparison with the reference pro-form molecule. When twoor more chemical modification (e.g. two proteolytic cleavages, or aproteolytic cleavage and a deglycosylation) can be distinguished enroute to the production of a mature molecule, the reference precursormolecule may be termed a “pre-pro-form” molecule.

A “property” can describe any characteristic, including a physical,chemical, or activity characteristic property of a protein or antibodyto be optimized. For example, in certain aspects, the predeterminedproperty, characteristic or activity to be optimized can be selectedfrom is selected from reduction of protein-protein aggregation,enhancement of protein stability, increased protein solubility,increased protein pH stability, increased protein temperature stability,increased protein solvent stability, increased selectivity, decreasedselectivity, introduction of glycosylation sites, introduction ofconjugation sites, reduction of immunogenicity, enhancement of proteinexpression, increase in antigen affinity, decrease in antigen affinity,change in binding affinity, change in immunogenicity, change incatalytic activity, pH optimization, or enhancement of specificity. An“optimized” property refers to a desirable change in a particularproperty in a mutant protein or antibody compared to a template proteinor antibody, respectively.

As used herein, the term “pseudorandom” refers to a set of sequencesthat have limited variability, such that, for example, the degree ofresidue variability at another position, but any pseudorandom positionis allowed some degree of residue variation, however circumscribed.

“Quasi-repeated units”, as used herein, refers to the repeats to bere-assorted and are by definition not identical. Indeed the method isproposed not only for practically identical encoding units produced bymutagenesis of the identical starting sequence, but also thereassortment of similar or related sequences which may divergesignificantly in some regions. Nevertheless, if the sequences containsufficient homologies to be reasserted by this approach, they can bereferred to as “quasi-repeated” units.

As used herein “random peptide library” refers to a set ofpolynucleotide sequences that encodes a set of random peptides, and tothe set of random peptides encoded by those polynucleotide sequences, aswell as the fusion proteins contain those random peptides.

As used herein, “random peptide sequence” refers to an amino acidsequence composed of two or more amino acid monomers and constructed bya stochastic or random process. A random peptide can include frameworkor scaffolding motifs, which may comprise invariant sequences.

As used herein, “receptor” refers to a molecule that has an affinity fora given ligand. Receptors can be naturally occurring or syntheticmolecules. Receptors can be employed in an unaltered state or asaggregates with other species. Receptors can be attached, covalently ornon-covalently, to a binding member, either directly or via a specificbinding substance. Examples of receptors include, but are not limitedto, antibodies, including monoclonal antibodies and antisera reactivewith specific antigenic determinants (such as on viruses, cells, orother materials), cell membrane receptors, complex carbohydrates andglycoproteins, enzymes, and hormone receptors.

“Recombinant” proteins refer to enzymes produced by recombinant DNAtechniques, i.e., produced from cells transformed by an exogenous DNAconstruct encoding the desired protein. “Synthetic” proteins are thoseprepared by chemical synthesis.

The term “related polynucleotides” means that regions or areas of thepolynucleotides are identical and regions or areas of thepolynucleotides are heterologous.

“Reductive reassortment”, as used herein, refers to the increase inmolecular diversity that is accrued through deletion (and/or insertion)events that are mediated by repeated sequences.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides: “reference sequence,” “comparisonwindow,” “sequence identity,” “percentage of sequence identity,” and“substantial identity.”

A “reference sequence” is a defined sequence used as a basis for asequence comparison; a reference sequence may be a subset of a largersequence, for example, as a segment of a full-length cDNA or genesequence given in a sequence listing, or may comprise a complete cDNA orgene sequence. Generally, a reference sequence is at least 20nucleotides in length, frequently at least 25 nucleotides in length, andoften at least 50 nucleotides in length. Since two polynucleotides mayeach (1) comprise a sequence (i.e., a portion of the completepolynucleotide sequence) that is similar between the two polynucleotidesand (2) may further comprise a sequence that is divergent between thetwo polynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a “comparison window” to identify and comparelocal regions of sequence similarity.

“Repetitive Index (RI)”, as used herein, is the average number of copiesof the quasi-repeated units contained in the cloning vector.

The term “saturation” refers to a technique of evolution wherein everypossible change is made at each position of a template polynucleotide ortemplate polypeptide; however the change at each position is notconfirmed by testing, but merely assumed statistically wherein themajority of possible changes or nearly every possible change isestimated to occur at each position of a template. Saturationmutagenesis refers to mutating the DNA of a region of a gene encoding aprotein that changes codon amino acid sequence of the protein and thenscreening the expressed mutants of essentially all of the mutants for animproved phenotype based on statistical over-sampling that approachescomprehensive coverage, but does not guarantee complete coverage.

The term “sequence identity” means that two polynucleotide sequences areidentical (i.e., on a nucleotide-by-nucleotide basis) over the window ofcomparison. The term “percentage of sequence identity” is calculated bycomparing two optimally aligned sequences over the window of comparison,determining the number of positions at which the identical nucleic acidbase (e.g., A, T, C, G, U, or I) occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison (i.e., thewindow size), and multiplying the result by 100 to yield the percentageof sequence identity. This “substantial identity”, as used herein,denotes a characteristic of a polynucleotide sequence, wherein thepolynucleotide comprises a sequence having at least 80 percent sequenceidentity, preferably at least 85 percent identity, often 90 to 95percent sequence identity, and most commonly at least 99 percentsequence identity as compared to a reference sequence of a comparisonwindow of at least 25-50 nucleotides, wherein the percentage of sequenceidentity is calculated by comparing the reference sequence to thepolynucleotide sequence which may include deletions or additions whichtotal 20 percent or less of the reference sequence over the window ofcomparison.

The term “silent mutation” refers to a codon change that does not resultin an amino acid change in an expressed polypeptide and is based onredundancy of codon usage for amino acid insertion.

As known in the art “similarity” between two proteins is determined bycomparing the amino acid sequence and its conserved amino acidsubstitutes of one protein to the sequence of a second protein.Similarity may be determined by procedures which are well-known in theart, for example, a BLAST program (Basic Local Alignment Search Tool atthe National Center for Biological Information).

As used herein, the term “single-chain antibody” refers to a polypeptidecomprising a VH domain and a VL domain in polypeptide linkage, generallyliked via a spacer peptide (e.g., [Gly-Gly-Gly-Gly-Ser]_(X)), and whichmay comprise additional amino acid sequences at the amino- and/orcarboxy-termini. For example, a single-chain antibody may comprise atether segment for linking to the encoding polynucleotide. As an examplea scFv is a single-chain antibody. Single-chain antibodies are generallyproteins consisting of one or more polypeptide segments of at least 10contiguous amino substantially encoded by genes of the immunoglobulinsuperfamily (e.g., see Williams and Barclay, 1989, pp. 361-368, which isincorporated herein by reference), most frequently encoded by a rodent,non-human primate, avian, porcine bovine, ovine, goat, or human heavychain or light chain gene sequence. A functional single-chain antibodygenerally contains a sufficient portion of an immunoglobulin superfamilygene product so as to retain the property of binding to a specifictarget molecule, typically a receptor or antigen (epitope).

The members of a pair of molecules (e.g., an antibody-antigen pair or anucleic acid pair) are said to “specifically bind” to each other if theybind to each other with greater affinity than to other, non-specificmolecules. For example, an antibody raised against an antigen to whichit binds more efficiently than to a non-specific protein can bedescribed as specifically binding to the antigen. (Similarly, a nucleicacid probe can be described as specifically binding to a nucleic acidtarget if it forms a specific duplex with the target by base pairinginteractions (see above).)

“Specific hybridization” is defined herein as the formation of hybridsbetween a first polynucleotide and a second polynucleotide (e.g., apolynucleotide having a distinct but substantially identical sequence tothe first polynucleotide), wherein substantially unrelatedpolynucleotide sequences do not form hybrids in the mixture.

The term “specific polynucleotide” means a polynucleotide having certainend points and having a certain nucleic acid sequence. Twopolynucleotides wherein one polynucleotide has the identical sequence asa portion of the second polynucleotide but different ends comprises twodifferent specific polynucleotides.

“Stringent hybridization conditions” means hybridization will occur onlyif there is at least 90% identity, preferably at least 95% identity andmost preferably at least 97% identity between the sequences. SeeSambrook et al., 1989, which is hereby incorporated by reference in itsentirety.

Also included in the invention are polypeptides having sequences thatare “substantially identical” to the sequence of a polypeptide, such asone of any SEQ ID NO disclosed herein. A “substantially identical” aminoacid sequence is a sequence that differs from a reference sequence onlyby conservative amino acid substitutions, for example, substitutions ofone amino acid for another of the same class (e.g., substitution of onehydrophobic amino acid, such as isoleucine, valine, leucine, ormethionine, for another, or substitution of one polar amino acid foranother, such as substitution of arginine for lysine, glutamic acid foraspartic acid, or glutamine for asparagine).

Additionally a “substantially identical” amino acid sequence is asequence that differs from a reference sequence or by one or morenon-conservative substitutions, deletions, or insertions, particularlywhen such a substitution occurs at a site that is not the active sitethe molecule, and provided that the polypeptide essentially retains itsbehavioural properties. For example, one or more amino acids can bedeleted from a phytase polypeptide, resulting in modification of thestructure of the polypeptide, without significantly altering itsbiological activity. For example, amino- or carboxyl-terminal aminoacids that are not required for phytase biological activity can beremoved. Such modifications can result in the development of smalleractive phytase polypeptides.

The present invention provides a “substantially pure protein”. The term“substantially pure protein” is used herein to describe a molecule, suchas a polypeptide (e.g., a phytase polypeptide, or a fragment thereof)that is substantially free of other proteins, lipids, carbohydrates,nucleic acids, and other biological materials with which it is naturallyassociated. For example, a substantially pure molecule, such as apolypeptide, can be at least 60%, by dry weight, the molecule ofinterest. The purity of the polypeptides can be determined usingstandard methods including, e.g., polyacrylamide gel electrophoresis(e.g., SDS-PAGE), column chromatography (e.g., high performance liquidchromatography (HPLC)), and amino-terminal amino acid sequence analysis.

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual macromolecular species in the composition),and preferably substantially purified fraction is a composition whereinthe object species comprises at least about 50 percent (on a molarbasis) of all macromolecular species present. Generally, a substantiallypure composition will comprise more than about 80 to 90 percent of allmacromolecular species present in the composition. Most preferably, theobject species is purified to essential homogeneity (contaminant speciescannot be detected in the composition by conventional detection methods)wherein the composition consists essentially of a single macromolecularspecies. Solvent species, small molecules (<500 Daltons), and elementalion species are not considered macromolecular species.

As used herein, “template oligopeptide” means a protein for which asecondary library of variants is desired. As will be appreciated bythose in the art, any number of templates find use in the presentinvention. Specifically included within the definition of “proteins” or“oligopeptides” are fragments and domains of known proteins, includingfunctional domains such as enzymatic domains, binding domains, etc., andsmaller fragments, such as turns, loops, etc. That is, portions ofproteins may be used as well. In addition, “protein” as used hereinincludes proteins, oligopeptides and peptides. In addition, proteinvariants, i.e., non-naturally occurring protein analog structures, maybe used.

Suitable proteins include, but are not limited to, industrial andpharmaceutical proteins, including ligands, cell surface receptors,antigens, antibodies, cytokines, hormones, transcription factors,signaling modules, cytoskeletal proteins and enzymes. Suitable classesof enzymes include, but are not limited to, hydrolases such asproteases, carbohydrases, lipases; isomerases such as racemases,epimerases, tautomerases, or mutases; transferases, kinases,oxidoreductases, and phophatases. Suitable enzymes are listed in theSwiss-Prot enzyme database. Suitable protein backbones include, but arenot limited to, all of those found in the protein data base compiled andserviced by the Research Collaboratory for Structural Bioinformatics(RCSB, formerly the Brookhaven National Lab).

As used herein, the term “variable segment” refers to a portion of anascent peptide which comprises a random, pseudorandom, or definedkernel sequence. A variable segment” refers to a portion of a nascentpeptide which comprises a random pseudorandom, or defined kernelsequence. A variable segment can comprise both variant and invariantresidue positions, and the degree of residue variation at a variantresidue position may be limited: both options are selected at thediscretion of the practitioner. Typically, variable segments are about 5to 20 amino acid residues in length (e.g., 8 to 10), although variablesegments may be longer and may comprise antibody portions or receptorproteins, such as an antibody fragment, a nucleic acid binding protein,a receptor protein, and the like.

The term “wild-type”, or wild type”, means that the polynucleotide doesnot comprise any mutations. A “wild type” protein means that the proteinwill be active at a level of activity found in nature and will comprisethe amino acid sequence found in nature.

DETAILED DESCRIPTION OF THE INVENTION

Currently, informatics are used up front to guide the evolution ofpolypeptide and protein molecules by deciding where in the molecules tomake mutations in a quest for molecular optimization. The disclosureprovides methods wherein mutations are systematically performedthroughout the polypeptide or protein first, then a map is created toprovide useful informatics at the back end and the map becomes the guidefor where to focus the next round of mutation. Various methods ofcomprehensive evolution are utilized alone and in combination in orderto provide highly predictive data for protein optimization.

The present invention relates to methods of identifying and mappingmutant polypeptides formed from, or based upon, a template polypeptide.These methods of evolution can be applied to all protein therapeutictypes such as, for example, hormones, enzymes, cytokines and antibodies.

Historically, discovery of antibodies has been performed in eukaryotic(euk) and prokaryotic (prok) hosts. Typically, in bacteria (E. coli),partial length antibodies are discovered; for example, in phage displaytechnologies, Fabs are recovered and sometimes converted to full lengthdownstream. There are several potential disadvantages to theseapproaches.

In one example, there is some evidence that Fc and Fv regionscommunicate to effect antibody properties, such as binding andexpression. Therefore, when an antibody fragment is optimized for aproperty such as expression, the improvement does not always translateto improved expression in the full length assembled antibody. Forexample, a library of Fc's was created in attempts to find a “holygrail” Fc that could be attached to any Fv to improve expression in anyhost.

In one aspect, codon mutagenesis was performed in the Constant Regionfor optimization of mammalian cell expression. Specifically 326 mutantswere created in the constant region and expressed in HEK 293 and CHO-Scells. Screening was performed by ELISA. Several Fc's met the criteriaof improved expression, and certain optimized Fc's were even identifiedthat transferred positive effects across multiple cell lines; however,when a different Fv was attached to the Fc, the improvement inexpression did not translate. This demonstrates that Fc's and Fv'scommunicate.

In order to avoid unexpected results upon recombination of antibodyfragments, in one preferred aspect, the methods of the disclosure areused to discover full length antibody molecules. In another preferredaspect, certain methods of the disclosure utilize euk hosts.

In one embodiment, the eukaryotic system is a mammalian system isselected from one of the group consisting of CHO, HEK293, IM9, DS-1,THP-1, Hep G2, COS, NIH 3T3, C33a, A549, A375, SK-MEL-28, DU 145, PC-3,HCT 116, Mia PACA-2, ACHN, Jurkat, MM1, Ovcar 3, HT 1080, Panc-1, U266,769P, BT-474, Caco-2, HCC 1954, MDA-MB-468, LnCAP, NRK-49F, and SP2/0cell lines; and mouse splenocytes and rabbit PBMC. In one aspect, themammalian system is selected from a CHO or HEK293 cell line. In onespecific aspect, the mammalian system is a CHO-S cell line. In anotherspecific aspect, the mammalian system is a HEK293 cell line. In anotherembodiment, the eukaryotic system is a yeast cell system. In one aspect,the eukaryotic system is selected from S. cerevisiae yeast cells orpicchia yeast cells.

In another embodiment, mammalian cell line creation can be performedcommercially by a contract research or custom manufacturingorganization. For example, for recombinant antibodies or other proteins,Lonza (Lonza Group Ltd, Basel, Switerland) can create vectors to expressproduct using the GS Gene Expression System™ technology with eitherCHOK1 SV or NSO host cell lines.

In another embodiment, evolution can be performed in prok hosts (such asE. coli) and screens can occur in euk hosts (for example, CHO).

Methods for evolving molecules include stochastic (random) andnon-stochastic methods. Published methods include random and non-randommutagenesis approaches. Any of these approaches can be employed toevolve properties of the therapeutic proteins of the present inventiontoward a desired characteristic, such as better stability in differenttemperature or pH environments, or better expression in a host cell.Other potentially desirable properties, such as improved catalyticactivity, improved protein stability in various conditions, improvedselectivity and/or solubility, and improved expression results byimprovement of characteristics such as reduced aggregation can beselected for in evolution experiments.

Evolution is performed directly in a eukaryotic host, such as amammalian cell host or a yeast cell host, that will be used fordownstream production of the therapeutic protein. Candidates can beevolved for optimal expression in the same host used to screen and/orevolve and to manufacture. Expression optimization can be achieved byoptimization of vectors used (vector components, such as promoters,splice sites, 5′ and 3′ termini and flanking sequences), genemodification of host cells to reduce gene deletions and rearrangements,evolution of host cell gene activities by in vivo or in vitro methods ofevolving relevant genes, optimization of host glycosylating enzymes byevolution of relevant genes, and/or by chromosome wide host cellmutagenesis and selection strategies to select for cells with enhancedexpression capabilities. Host cells are further described herein.

Cell surface display expression and screening technology (for example,as defined above) can be employed to screen libraries of evolvedproteins for candidates to be manufactured.

Current methods in widespread use for creating alternative proteins froma starting molecule are oligonucleotide-directed mutagenesistechnologies, error-prone polymerase chain reactions and cassettemutagenesis, in which the specific region to be optimized is replacedwith a synthetically mutagenized oligonucleotide. In these cases, anumber of mutant sites are generated around certain sites in theoriginal sequence.

In oligonucleotide-directed mutagenesis, a short sequence is replacedwith a synthetically mutagenized oligonucleotide. Error-prone PCR useslow-fidelity polymerization conditions to introduce a low level of pointmutations randomly over a long sequence. In a mixture of fragments ofunknown sequence, error-prone PCR can be used to mutagenize the mixture.In cassette mutagenesis, a sequence block of a single template istypically replaced by a (partially) randomized sequence.

Chimeric genes have been made by joining 2 polynucleotide fragmentsusing compatible sticky ends generated by restriction enzyme(s), whereeach fragment is derived from a separate progenitor (or parental)molecule. Another example is the mutagenesis of a single codon position(i.e. to achieve a codon substitution, addition, or deletion) in aparental polynucleotide to generate a single progeny polynucleotideencoding for a single site-mutagenized polypeptide.

Further, in vivo site specific recombination systems have been utilizedto generate hybrids of genes, as well as random methods of in vivorecombination, and recombination between homologous but truncated geneson a plasmid. Mutagenesis has also been reported by overlappingextension and PCR.

Non-random methods have been used to achieve larger numbers of pointmutations and/or chimerizations, for example comprehensive or exhaustiveapproaches have been used to generate all the molecular species within aparticular grouping of mutations, for attributing functionality tospecific structural groups in a template molecule (e.g. a specificsingle amino acid position or a sequence comprised of two or more aminoacids positions), and for categorizing and comparing specific groupingof mutations. U.S. Pat. No. 7,033,781 entitled “Whole cell engineeringmy mutagenizing a substantial portion of a starting genome, combiningmutations, and optionally repeating” describes a method of evolving anorganism toward desired characteristics. U.S. Pat. No. 6,764,835entitled “Saturation mutagenesis in directed evolution” and U.S. Pat.No. 6,562,594 entitled “Synthetic ligation reassembly in directedevolution” describe methods of exhaustively evolving and screening fordesired characteristics of molecules. Any such methods can be used inthe method of the present invention.

There is a difference between previously known methods of “saturationmutagenesis” and techniques of “comprehensive” evolution preferredherein. Saturation mutagenesis refers to a technique of evolutionwherein every possible change is made at each position of a templatepolynucleotide or template polypeptide; however the change at eachposition is not confirmed by testing, but merely assumed statistically.Comprehensive evolution refers to a technique of evolution wherein everypossible change is made at each position of a template polynucleotide ortemplate polypeptide and the polynucleotide or polypeptide is tested toconfirm the intended change has been made.

Saturation methods are inherently statistical, non-comprehensive methodsand were also never truly comprehensive across all the steps (forexample, across mutant generation, mutant identification, mutant proteinexpression, mutant protein screening, and recombined up-mutantgeneration, identification, expression and screening). In comprehensiveevolution techniques, each molecule is screened and confirmed at boththe first step of mutagenesis, and further at a second step ofrecombining the up-mutants or hits.

Unless the saturation mutagenesis is confirmed by sequencing or someother method, the technique cannot be considered to be comprehensive forseveral possible reasons. For example, 1) cloning systems are not 100%efficient due to due to cloning or synthesis errors, or difficult toclone molecules or 2) some proteins are toxic when expressed and thuscannot be efficiently expressed. Therefore, it is important to confirmby sequencing, or some other technique, at each step. It is useful toscore every step in order to screen for expression, so non-expressingclones don't get designated as “negative” as in previous work, they justget scored non-expressible. Comprehensive techniques are thereforeconsidered to be more pure non-stochastic system than saturationtechniques, as confirmed by the “confirmation” step.

Comprehensive Positional Evolution

Referring to FIG. 1, using a linear peptide as a simple example, in afirst step, a set of naturally occurring amino acid variants (or asubset thereof, or amino acid derivatives) for each codon from position1 to n (n corresponding to the number of residues in the polypeptidechain) is generated by a process referred to herein as ComprehensivePositional Evolution (CPE™). This procedure is repeated for eachpolypeptide chain of the target molecule. A minimum set of amino acidmutations contains only one codon for each of the 19 natural aminoacids. However, it is recognized that each expression system may sufferfrom codon bias, in which insufficient tRNA pools can lead totranslation stalling, premature translation termination, translationframeshifting and amino acid misincorporation. Therefore, for expressionoptimization each set contains up to 63 different codons, including stopcodons. In the next step, the mutations are confirmed by sequencing eachnew molecule. Other methods of confirmation can also be employed.

Each amino acid set is then screened for at least one of:

-   -   Improved function    -   Neutral mutations    -   Inhibitory mutations    -   Expression    -   Compatibility of the clone with the host system.

In one aspect, multiple characteristics are screened for simultaneouslysuch as, for example, improved function and expression.

The data for each set are combined for the entire polypeptide chain(s)and a detailed functional map (referred to herein as an EvoMap™) of thetarget molecule is generated. This map contains detailed information howeach mutation affects the performance/expression and/or cloningcapability of the target molecule. It allows for the identification ofall sites where no changes can be made without a loss in proteinfunction (or antigen/receptor binding in case of antibodies). It alsoshows where changes can be made without affecting function. The mapfurther identifies changes that result in molecules that do not expressin the host system, and therefore do not assess the effect of themutation.

A schematic of a hypothetical EvoMap™ is shown in FIG. 1. Each positionon the template is identified as a restricted site (non-mutable), afully mutable site, a partially mutable site or an up-mutant for aspecific amino acid substitution. Each partially mutable site may befurther designated as amenable to substitution with, for example, acharged residue, or a non-polar residue substitution, and anon-expressing clone and/or molecule that cannot be cloned in the hostsystem.

It is possible to utilize the EvoMap™ in order to recognize andrecombine beneficial single amino acid substitutions, and screen tofurther optimize the desired characteristics in the target molecule.However, evolution of certain characteristics may require two or moresimultaneous mutations to become observable. The EvoMap™ may beexploited to efficiently, and cost effectively, produce a set ofmulti-site mutant polypeptides in a non-random fashion. The set ofmulti-site mutant polypeptides can then be screened for multi-siteupmutants.

CPE enables the complete in vivo confirmed protein mutation map.Identification of the entire set of up-mutants enables furthercombinatorial evolution step(s). CPE can be utilized in order to reducethe immunogenicity risk of evolved proteins by the selection ofnon-surface mutations; elimination of T-cell epitopes; and mimicry ofsomatic mutations.

In one aspect, CPE can be used to generate a library of up to 5, 10 or15 amino acids, or up to all 19 amino acids. Changes are made at eachposition in the protein and screened for a desirable characteristic,such as binding affinity or expression, and the Evomap™ is created.Later rounds of mutation and screening can be used to generate the datafor all 19 amino acids. From the map, fully mutable sites areidentified. These sites are useful to identify positions that can bemodified to create a new collection of molecules that can be made andtested for new characteristics. For example, informatics can be employedto identify HLA haplotypes in the sequence, and desired changes can bemade to avoid these haplotypes by making specific targeted changes at“neutral” (“fully mutable”) sites identified from the map, where theprimary characteristic will not be affected. This could potentiallyreduce immunogenicity risk (one could select non-surface mutations,eliminate t-cell epitopes, mimic hypersomatic mutations). Further, themap can show sites for site specific modifications (glycosylation andchemical conjugation) to improve various characteristics. Also,optimization of silent mutations can improve protein expression in avariety of hosts.

Synergy Evolution

In one embodiment of the present invention, an EvoMap™ is generated andutilized for Synergy Evolution, as shown in FIG. 2. In SynergyEvolution, simultaneous mutation at 2-20 selected sites may be combinedto produce a combinatorial effect. The EvoMap™ of the templatepolypeptide is used to select specific single amino acid point mutationsfor assembly to multi-site polypeptide mutations.

In Synergy Evolution, non-deactivating amino acid point mutations areselected from within partially mutable sites that are near non-mutablesites on the EvoMap™. In one aspect, the selected non-deactivating pointmutations are adjacent to non-mutable sites. In Synergy Evolution,simultaneous mutation of amino acids at two to 20 of the selected sitesis performed for combinatorial effects. In one aspect, recombination oftwo to 20 selected mutations is used to produce a codon variant librarycoding for a population of multi-site mutant polypeptides. In oneaspect, the mutations are confirmed by sequencing each new molecule.Other methods of confirmation can also be employed.

Following cloning and expression, the multi-site mutant polypeptidesproduced are then screened for at least one predetermined property,characteristic or activity compared to the template polypeptide. In thismanner, multi-site upmutant polypeptides can be identified. In oneaspect, multi-site mutant polypeptides are produced by combinatorialprotein synthesis. One advantage of Synergy Evolution is that it doesnot require a protein x-ray crystal structure to direct evolution of thetemplate polypeptide. This technique is useful particularly for proteinswith high assay variation and other multi-site effects.

According to the present invention, applications of Synergy Evolutioninclude, but are not limited to evolution of complex molecularmechanistic changes, evolution of proteins with high assay variation,evolution of protein specificity, improvement of expression in variousexpression hosts, improvement of protein catalytic activity, stability,and pH optimization. Synergy Evolution is applicable to all proteintherapeutic types including, but not limited to, hormones, enzymes,cytokines and antibodies.

In one aspect of the present invention, Synergy Evolution can be used tooptimize one or more aspects of a polypeptide which is a portion of aprotein molecule. The protein molecule can be assembled by ligating oneor more mutant nucleic acids coding for polypeptides with zero, one ormore nucleic acids coding for framework polypeptides to create a variantprotein by cloning, translation and expression techniques known in theart. In one aspect, a framework polypeptide is derived from a wild-typeprotein molecule. In this aspect, Synergy Evolution can be used inconjunction with antibody humanization techniques. For example, a mousemonoclonal antibody may be selected for evolution and humanization. TheCDR regions of the antibody are cloned and sequenced and individual CDRregions (CDR1, CDR2, CDR3) may be synthesized and ligated to othernnucleotides coding for human antibody framework polypeptides, followedby prodcution of a human variant IgG library. The human variant IgGlibrary is then screened for at least one property compared to the mousemAb. In another aspect, a framework polypeptide is an artificialscaffold polypeptide. Specific techniques of ds DNA fragmentpreparation, ligation and assembly of nucleic acids, cloning,transfection, expression, solid phase synthesis of libraries, solutionphase synthesis of libraries, comprehensive positional evolution,combinatorial protein synthesis, quantification of expression by ELISAquantification and β-galactosidase assay, and functional ELISA arepresented in the examples section.

In another embodiment of the invention, Synergy Evolution can be used toenhance binding affinity of an antibody. In this embodiment,optimization of the antibody variable region may be performed. Forexample, for the production of antibody mutants, CPE is performed forlight chain and heavy chain variable regions of a selected antibody andan EvoMap™ is generated. Mutants are selected for reassembly; forexample, variants of the light chain are selected and variants of theheavy chain are selected for assembly. Non-deactivating amino acid pointmutations are selected from within partially mutable sites that are nearnon-mutable sites. The reassembly technology utilizing CPS can be usedto create a library of heavy chains. The light chain variants can becombined with the heavy chain variants, cloned, expressed and thevariants are screened as full IgGs from mammalian cell linesupernatants. Binding affinity for certain variants is assessed by, forexample, use of ELISA, BIAcore and/or Sapidyne instrumentation assays,or other techniques known to one in the art.

Flex Evolution

In another embodiment, the CPE/EvoMap may be used to identify andexploit fully mutable sites. In one aspect, exploitation of multiplefully mutable sites is termed Flex Evolution and is used to maketargeted changes such as introduction of sites for glycosylation (e.g.codons for amino acids for N- or O-linked glycosylation; Asn withinconsensus sequence Asn-Aa-Ser-Thr or Ser/Thr) and chemical conjugation.Flex evolution may also be used in design of protease cleavage sites,introduction of tags for purification and/or detection, site-specificlabeling, and the like. Further, codon optimization of silent mutationsmay be utilized for improvement of protein expression. In thisembodiment, termed Flex Evolution, following protein expression, themutant polypeptide libraries produced are rescreened for at least onepredetermined property, characteristic or activity compared to thetemplate polypeptide. In one aspect, the predetermined property includesreduction of protein-protein aggregation, enhancement of proteinstability, or increased protein solubility. In another aspect, anyexpression system which glycosylates may be used for the introduction ofglycosylation sites, such as, for example, mammalian, plant, yeast, andinsect cell lines.

In Flex Evolution, evaluation of bioinformatics and protein x-raycrystal structures of related proteins, or the template protein orpolypeptide, is useful for template optimization. In one aspect,selected sites are not at contact residues. In another aspect, selectionof non-surface protein mutations allows for reduced immunogenicity risk.

Applications of Flex Evolution include, bit are not limited to,reduction of protein-protein aggregation, improvement of proteinsolubility, optimization of pharmacokinetics via glycosylationlibraries, optimization of protein secondary and tertiary structure anddeimmunization of antigenic sites directly via either mutation sets orindirectly through glycosylation masking.

In one aspect of Flex Evolution, an EvoMap™ is utilized to identifyfully mutable sites, CPS generation is performed with insertion ofglycosylating residues to fully mutable sites (or silent mutations fortranslation effects), and screening of combinatorial glycosylatedlibrary is performed by analytical analysis (e.g. Mass Spectroscopyanalysis, Dynamic Light Scattering), immunogenicity reduction (bybioinformatics or assay), and/or pharmacokinetic analysis (e.g. inFoxnlnu mice).

In one aspect, Flex evolution may be used for deimmunization toeliminate immunogenicity while maintaining function. Flex Evolutiondeimmunization can be performed by masking immunogenicity withglycosylation, identifying human hypersomatic mutation spectra aminoacid substitutions that may eliminate immunogenicity while maintainingfunction, reduction of dose for evading immunogenicity potential, andminimization of non-surface amino acid residue changes. Further,immunogenicity databases and algorithms can be used to identify andreplace potential MHC binding epitopes. In one aspect, in silicomodification prediction is coupled with CPE or CPE combined with CPSdata to generate variants. In one aspect, the mutations are confirmed bysequencing each new molecule. Other methods of confirmation can also beemployed.

Reduced propensity to generate T-cell epitopes and/or deimmunization maybe measured by techniques known in the art. Preferably, deimmunizationof proteins may be tested in vitro by T cell proliferation assay. Inthis assay PBMCs from donors representing >80% of HLA-DR alleles in theworld are screened for proliferation in response to either wild type ordeimmunized peptides. Ideally cell proliferation is only detected uponloading of the antigen-presenting cells with wild type peptides.Additional assays for deimmunization include human in vitro PBMCre-stimulation assays (e.g. interferon gamma (TH1) or IL4 (TH2) ELISA.Alternatively, one may test deimmunization by expressing HLA-DRtetramers representing all haplotypes. In order to test if de-immunizedpeptides are presented on HLA-DR haplotypes, binding of e.g.fluorescence-labeled peptides on PBMCs can be measured. Measurement ofHLA Class I and Class II transgenic mice for responses to target antigen(e.g. interferon gamma or IL4). Alternatively epitope library screeningwith educated T cells (MHCl 9mer; MHCII 20mer) from PBMC and/ortransgenic mouse assays. Furthermore, deimmunization can be proven bydetermining whether antibodies against the deimmunized molecules havebeen generated after administration in patients.

In another embodiment, the Flex Evolution techniques of the presentinvention can be utilized for expression optimization. In one aspect,the present invention discloses the utilization of protein engineeringmethods to develop silent mutation codon optimized Fc variants withimproved expression in mammalian cells. A silent mutation is one inwhich the variation of the DNA sequence does not result in a change inthe amino acid sequence of the preotein. In one aspect, codonmutagenesis is performed in the constant region for optimization ofmammalian cell expression. A codon optimized Fc variant with improvedexpression properties while retaining the capacity to mediate effectorfunctions improves the production of therapeutic antibodies. In thisaspect, for example, a constant region of an antibody molecule can beevolved for screening in different expression hosts, for example,mammalian cell lines expression screening utilizing CHO, HEK293 andCOS-7. One example of expression optimization by codon mutagenesis inthe constant region for mammalian cell expression is shown in FIG. 3.The expression levels shown are each an average of 4 data points, andconfirmed over multiple experiments. Multiple cell line capability wasdemonstrated for first mutant tested in HEK293 and CHO cell lineexpression systems.

In addition, the EvoMap™ may be used to generate 3-dimensionalcomputational molecular models of the oligopeptide, or specific regionsthereof, to explore the structural mechanisms involved in, e.g.,antibody-epitope specificity and stability. A hypotheticalthree-dimensional EvoMap™ is shown in FIG. 9.

The information in EvoMap can also be combined with structuralinformation (if available) to select e.g., only surface residues formutations to increase solubility/decrease aggregation.

Comprehensive Positional Insertion Evolution

In one embodiment, the disclosure provides methods of identifying andmapping mutant polypeptides formed from, or based upon, a templatepolypeptide. Referring to FIG. 4, using a linear peptide as a simpleexample, in a first step, a set of naturally occurring amino acidvariants (or a subset thereof, or amino acid derivatives) for each codonfrom position 1 to n (n corresponding to the number of residues in thepolypeptide chain) is generated by a process referred to herein asComprehensive Positional Insertion (CPI™) evolution.

In CPI™, an amino acid is inserted after each amino acid throughout atemplate polypeptide one at a time to generate a set of lengthenedpolypeptides. CPI can be used to insert 1, 2, 3, 4, or up to 5 new sitesat a time. Each of the 20 amino acids is added at each new position, oneat a time, creating a set of 20 different molecules at each new positionadded in the template. In this case, position 1, which is methionine andinvariant, is skipped. This procedure is repeated for each polypeptidechain of the target molecule. A minimum set of amino acid mutationscontains only one codon for each of the 20 natural amino acids. In oneaspect, the mutations are confirmed by sequencing each new molecule.Other methods of confirmation can also be employed.

The present invention relates to methods of identifying and mappingmutant polypeptides formed from, or based upon, a template polypeptide.Typically, the polypeptide will comprise n amino acid residues, whereinthe method comprises (a) generating n+[20×(n−1)] separate polypeptides,wherein each polypeptide differs from the template polypeptide in thatit has inserted after each position in the template each of the 20 aminoacids one at a time (as illustrated in FIG. 1); confirming the changesby sequencing or some other technique; assaying each polypeptide for atleast one predetermined property, characteristic or activity; and (b)for each member identifying any change in said property, characteristicor activity relative to the template polypeptide

In one embodiment, one or more regions are selected for mutagenesis toadd one position at a time as described above. In such case, nrepresents a subset or region of the template polypeptide. For example,where the polypeptide is an antibody, the entire antibody or one or morecomplementarity determining regions (CDRs) of the antibody are subjectedto mutagenesis to add one position at a time in the template polypeptideafter each position.

The invention thus includes methods of mapping a set of mutantantibodies formed from a template antibody having at least one, andpreferably six, complementarity determining regions (CDRs), the CDRstogether comprising n amino acid residues, the method comprising (a)generating n+[20×(n−1)] separate antibodies, wherein each antibodydiffers from the template antibody in that has inserted a singlepredetermined position, one at a time, after each position in thetemplate sequence; (b) assaying each set for at least one predeterminedproperty, characteristic or activity; and (c) for each memberidentifying any change in a property, characteristic or activityrelative to the template polypeptide. For antibodies, the predeterminedproperty, characteristic or property may be binding affinity and/orimmunogenicity, for example.

In addition, provided are methods of producing a set of mutantantibodies formed from a template antibody having at least onecomplementarity determining region (CDR), the CDR comprising n aminoacid residues, the method comprising: (a) generating n+[20×(n−1)]separate antibodies, wherein each antibody differs from the templateantibody in that it has an extra amino acid added at a singlepredetermined position of the CDR. In another embodiment, the antibodycomprises six CDRs, and together the CDRs comprise n amino acidresidues.

In another embodiment, the new lengthened polypeptides described aboveare further mutated and mapped after screening to identify a change in aproperty, characteristic or activity relative to the shortenedpolypeptide. Typically, the lengthened polypeptide will comprise n aminoacid residues, wherein the method comprises (a) generating n (n−1 in thecase where the initial residue is methionine) separate sets ofpolypeptides, each set comprising member polypeptides having X number ofdifferent predetermined amino acid residues at a single predeterminedposition of the polypeptide; wherein each set of polypeptides differs inthe single predetermined position; assaying each set for at least onepredetermined property, characteristic or activity; (b) for each memberidentifying any change in said property, characteristic or activityrelative to the template polypeptide; and optionally (c) creating afunctional map reflecting such changes. Preferably, the number ofdifferent member polypeptides generated is equivalent to n×X (or[n−1]×X, as the case may be).

In the alternative, the method comprises generating a single populationcomprising the sets of mutated polypeptides from the lengthenedpolypeptides. In this embodiment, the entire new population is screened,the individual members identified, and the functional map generated.

Typically, where each naturally occurring amino acid is used, X will be19 (representing the 20 naturally occurring amino acid residues andexcluding the particular residue present in a given position of thetemplate polypeptide). However, any subset of amino acids may be usedthroughout, and each set of polypeptides may be substituted with all ora subset of the total X used for the entire population.

However, it is recognized that each expression system may suffer fromcodon bias, in which insufficient tRNA pools can lead to translationstalling, premature translation termination, translation frameshiftingand amino acid misincorporation. Therefore, for expression optimizationeach set contains up to 61 different codons.

Each amino acid set is then screened for at least one desirablecharacteristic such as improved function; neutral mutations, inhibitorymutations, and expression.

In one aspect, the lengthened polypeptides can be mapped to identify achange in a property, characteristic or activity resulting in theshortened polypeptides relative to the “wildtype”. The data for each setare combined for the entire polypeptide, or “target molecule”. Hits fromthe screening of the lengthened polypeptides (target molecules) can thenbe used for further comprehensive mutagenesis chain(s) and screening asdescribed herein. The data from mutagenesis provides a detailedfunctional map (referred to herein as an EvoMap™) of the target moleculeis generated. This map contains detailed information how each mutationaffects the performance/expression of the target molecule. It allows forthe identification of all sites where no changes can be made without aloss in protein function (or antigen/receptor binding in case ofantibodies). It also shows where changes can be made without affectingfunction.

Comprehensive Positional Deletion Evolution

Comprehensive Positional Deletion Evolution (CPD™) relates to methods ofidentifying and mapping mutant polypeptides formed from, or based upon,a template polypeptide. CPD evolution deletes every amino acid throughthe protein one position at a time. Typically, the polypeptide willcomprise n amino acid residues, wherein the method comprises (a)generating n−1 (n−2 in the case where the initial residue is methionine)separate polypeptides, wherein each polypeptide differs from thetemplate polypeptide in that it lacks a single predetermined position;confirming the changes by sequencing or some other technique; assayingeach polypeptide for at least one predetermined property, characteristicor activity; and (b) for each member identifying any change in saidproperty, characteristic or activity relative to the templatepolypeptide.

In one embodiment of CPD evolution, one or more regions are selected formutagenesis to remove one position at a time. In such case, n representsa subset or region of the template polypeptide. For example, where thepolypeptide is an antibody, the entire antibody or one or morecomplementarity determining regions (CDRs) of the antibody are subjectedto mutagenesis to remove one position at a time in the templatepolypeptide. In one aspect, the mutations are confirmed by sequencingeach new molecule. Other methods of confirmation can also be employed.

In one embodiment, CPD thus includes methods of mapping a set of mutantantibodies formed from a template antibody having at least one, andpreferably six, complementarity determining regions (CDRs), the CDRstogether comprising n amino acid residues, the method comprising (a)generating (n−1) separate antibodies, wherein each antibody differs fromthe template antibody in that lacks a single predetermined position; (b)assaying each set for at least one predetermined property,characteristic or activity; and (c) for each member identifying anychange in a property, characteristic or activity relative to thetemplate polypeptide. For antibodies, the predetermined property,characteristic or property may be binding affinity and/orimmunogenicity, for example.

One aspect of CPD evolution includes methods of producing a set ofmutant antibodies formed from a template antibody having at least onecomplementarity determining region (CDR), the CDR comprising n aminoacid residues, the method comprising: (a) generating n−1 separateantibodies, wherein each antibody differs from the template antibody inthat lacks a single predetermined position of the CDR. In anotherembodiment, the antibody comprises six CDRs, and together the CDRscomprise n amino acid residues.

In another embodiment of CPD evolution, the new shortened polypeptidesdescribed above are further mutated and mapped after screening toidentify a change in a property, characteristic or activity relative tothe shortened polypeptide. Typically, the shortened polypeptide willcomprise n amino acid residues, wherein the method comprises (a)generating n (n−1 in the case where the initial residue is methionine)separate sets of polypeptides, each set comprising member polypeptideshaving X number of different predetermined amino acid residues at asingle predetermined position of the polypeptide; wherein each set ofpolypeptides differs in the single predetermined position; assaying eachset for at least one predetermined property, characteristic or activity;(b) for each member identifying any change in said property,characteristic or activity relative to the template polypeptide; and (c)creating a functional map reflecting such changes. Preferably, thenumber of different member polypeptides generated is equivalent to n×X(or [n−1]×X, as the case may be).

In the alternative, the CPD method comprises generating a singlepopulation comprising the sets of mutated polypeptides from theshortened polypeptides. In this embodiment, the entire new population isscreened, the individual members identified, and the functional mapgenerated. Typically, where each naturally occurring amino acid is used,X will be 19 (representing the 20 naturally occurring amino acidresidues and excluding the particular residue present in a givenposition of the template polypeptide). However, any subset of aminoacids may be used throughout, and each set of polypeptides may besubstituted with all or a subset of the total X used for the entirepopulation.

Any mutational or synthetic means may be used to generate the set ofmutants in CPD evolution. In one embodiment, the generation ofpolypeptides comprises (i) subjecting a codon-containing polynucleotideencoding for the template polypeptide to polymerase-based amplificationusing a 64-fold degenerate oligonucleotide for each codon to bemutagenized, wherein each of the 64-fold degenerate oligonucleotides iscomprised of a first homologous sequence and a degenerate N,N,N tripletsequence, so as to generate a set of progeny polynucleotides; and (ii)subjecting the set of progeny polynucleotides to clonal amplificationsuch that polypeptides encoded by the progeny polynucleotides areexpressed.

In one embodiment of CPD evolution, the entire shortened polypeptide issubjected to comprehensive mutagenesis. In another embodiment, one ormore regions are selected for comprehensive mutagenesis. In such case, nrepresents a subset or region of the template polypeptide. For example,where the polypeptide is an antibody, the entire antibody or one or morecomplementarity determining regions (CDRs) of the antibody are subjectedto comprehensive mutagenesis.

The CPD evolution disclosure thus includes methods of mapping a set ofmutant antibodies formed from a shortened template antibody having atleast one, and preferably six, complementarity determining regions(CDRs), the CDRs together comprising n amino acid residues, the methodcomprising (a) generating n separate sets of antibodies, each setcomprising member antibodies having X number of different predeterminedamino acid residues at a single predetermined position of the CDR;wherein each set of antibodies differs in the single predeterminedposition; and the number of different member antibodies generated isequivalent to n×X; (b) assaying each set for at least one predeterminedproperty, characteristic or activity; (c) for each member identifyingany change in a property, characteristic or activity relative to thetemplate polypeptide; and (d) creating a structural positional map ofsuch changes. For antibodies, the predetermined property, characteristicor property may be binding affinity and/or immunogenicity. As set forthabove, in the alternative a single population comprising all sets ofmutated antibodies may be generated.

In addition, provided are methods of producing a set of mutantantibodies formed from a shortened template antibody having at least onecomplementarity determining region (CDR), the CDR comprising n aminoacid residues, the method comprising: (a) generating n separate sets ofantibodies, each set comprising member antibodies having X number ofdifferent predetermined amino acid residues at a single predeterminedposition of the CDR; wherein each set of antibodies differs in thesingle predetermined position; and the number of different memberantibodies generated is equivalent to n×X. In another embodiment,antibody comprises six CDRs, and together the CDRs comprise n amino acidresidues.

The CPD™ evolution method includes a functional positional map (EvoMap™)made by the methods described herein. In an additional embodiment,certain residues particularly sensitive to change may be so indicated onthe EvoMap™. Further optimization may be implemented by makingadditional mutational changes at positions outside of these sensitivepositions. It is also possible to utilize the EvoMap™ in order torecognize and recombine beneficial single amino acid substitutions, andscreen to further optimize the desired characteristics in the targetmolecule, in a process called Combinatorial Protein Synthesis (CPS™)

Combinatorial Protein Synthesis

Combinatorial Protein Synthesis (CPS™) involves combining individualhits from CPE, CPI, CPD, or any other evolutionary technique tosynthesize proteins with combined mutations which are then screened foroptimized gene and protein characteristics. Ususually up-mutants orneutral mutations from other techniques of evolution are combined inCPS. A schematic of CPS is shown in FIG. 8. Comprehensive CPS refers totaking all of the theoretical selected up-mutants and generating allcombinations and sequencing them all prior to activity/expressionscreening to ensure that the clones exist in the set and to determinewhether they can be expressed in the system. In essentially everyprotein there will be mutants that express insufficient levels foractivity detection and these need to be scored for Comprehensive ProteinSynthesis and screening. i.e. CPS process.

In one embodiment CPE is followed by CPS to create mutants, which arescreened for the desired property. In one aspect, time and resources canbe saved in the CPE process by changing 2aa or 3 aa or 4 aas at a timeversus one at a time; so if the number of aa's in the protein is N, thetotal number generated and screened for 2 aa at a time would be (20²)×½;3 at a time would be (20³)×⅓, etc. For example, in one specific aspect,(in the 2aa example): 1^(st) aa at 1^(st) aa position is combined withall 20 at the 2^(nd) aa position and all the other aa's remain the same,then the 2^(nd) aa at 1^(st) aa position is combined with all 20 at the2^(nd) aa position and all other aa's remain the same. The entirepopulation is screened for up mutants and then mutation at the secondset of the next two aa's down the line is performed. In a similaraspect, this can be performed for 3aas at a time or 4aas at a time. Inanother aspect, optionally follow the CPE process with CPS of upmutants(including any subset thereof).

In one aspect, non-natural amino acids can be incorporated into theprocess (so all 19 other amino acids, or a subset thereof, plusnon-natural amino acids) by using novel technologies such as thequadruplet codon described in the attached and related papers. Neumannet al. Encoding multiple unnatural amino acids via evolution of aquadruplet-decoding ribosome Nature 464, 441-444 (14 Feb. 2010). In thisaspect CPE or CPE combined with CPS is performed for incorporation ofnon-natural amino acids. In a another aspect, informatics can beutilized after CPE or CPE combined with CPS to add further natural ornon-natural amino acids.

In a further aspect, the entire CPE library is created synthetically(synthesizing all the molecules on commercially available machines). Inthe event the synthesis machine cannot create large enough strands,fragments are synthesized and then ligated to generate full lengthmolecules. This library is screened and followed with CPS to combinedesired mutations. This is a two step process wherein CPE is followed byCPS, not one step of only CPE.

In another aspect, a CPE library is generated and screened, thenfollowed by CPS combining up mutants as follows: if there are 10up-mutants, test a single molecule with all 10 changes, then test allversions of 9 mutations, then 8, 7, 6, 5 etc. until one of the groupsdoesn't find an improved molecule over any in the previous group. Oncean improved molecule is identified the process can be terminated.

In a further aspect, CPE is performed to identify up-mutants and neutralmutations for affinity and expression, then CPS is performed withcombinations of up mutants and neutral mutations, and the library isrescreened for further improvements in characteristics such as function,affinity and/or expression.

In a further aspect, CPE is performed on codons of the Fc or otherdomain for glycosylation changes.

In another aspect, CPE or CPE combined with CPS of microRNA's or intronscan be performed.

In a further aspect, CPE or CPE combined with CPS of rodent antibodyCDRs is performed, then screened for up-mutants, followed byhumanization.

In one aspect, CPE or CPE combined with CPS is performed to producealternative intermediate nucleotides that lead to the desired mutationin the final reaction, for example, a methylated cytosine that convertsto a uracil.

In one aspect, CPE or CPE combined with CPS plus informatics is utilizedfor converting mouse CDR's to human CDR's and vice versa.

In one aspect, CPE or CPE combined with CPS is utilized with 2 and 3mutations spaced throughout the protein.

In another aspect, CPE or CPS combined with CPS are performed on heavychains and light chains in a dual chain vector for screening evaluationfor increased sensitivity.

In a further aspect, CPE or CPE combined with CPS is performed andmolecules are screened for selecting for allosteric changes in amolecule.

In one aspect, any of the evolution techniques of the disclosure cancomprise chemical synthesis of oligonucleotides. For example, IntegratedDNA Technologies (Coralville, Iowa) can synthesize high fidelityoligonucleotides known as “ULTRAmers™” of up to 200 bases in length, andup to 300mers, with quality control confirmation utilizing ESI-LC-MStechnology.

Any of several screening techniques can be used to evaluate CPE or CPEcombined with CPS mutants. In one aspect CPE or CPE combined with CPSmutants can be secreted and displayed in mammalian hosts. Alternatively,CPE or CPE combined with CPS mutants can be produced in E. coli andscreened in mammalian hosts. In another aspect, CPE is performedstarting at 15aa or 10aa and followed by CPS; then followed up with therest of the remaining 19aa. In another aspect, CPE or CPE combined withCPS is utilized for evolving proteins specifically with non-surfaceamino acid changes. In one aspect, CPE for can be utilized formulti-dimensional epitope mapping. In another aspect, CPE or CPEcombined with CPS screening can be performed transiently in mammaliancells. In a preferred aspect, CPE is performed, then sequencing andarray of all clones is performed, for example, in a chip based orwell-based format for expression and screening. In another aspect, CPEor CPE combined with CPS is utilized for evolving metal ion coordinationby selecting in varying ion concentrations. In a further aspect, CPE orCPE combined with CPS is performed, and proteins are expressed andscreened in cell free conditions and in non-human living organisms. Inone aspect, CPE or CPE combined with CPS screening of stem cells isperformed for varying effects on differentiation and protein and RNA andmRNA expression. In a further aspect, CPE or CPE combined with CPSmultiplex screening is performed for multiple protein characteristicslike expression and binding. In another aspect, CPE or CPE combined withCPS is performed on template molecules involved in brain transport andmembrane crossing; and mutants are screened for improvedcharacteristics. In one aspect, CPE or CPE combined with CPS mutants arescreened for protein hygroscopic characteristics. In another aspect, CPEor CPE combined with CPS mutants are assayed for selecting dynamicproteins. In one aspect, CPE or CPE combined with CPS screening isperformed outside of the target condition to identify mutants withintarget condition and vice versa.

In one embodiment, any of the above aspects of CPE or CPE combined withCPS are utilized in combination with a method selected from CPI, CPD,and CPD with CPI combination.

In another embodiment, any of the above aspects of CPE or CPE combinedwith CPS are utilized in combination with a method selected from Flexevolution and Synergy evolution performed from a template.

The term “template” may refer to a base polypeptide or a polynucleotideencoding such polypeptide. As would be appreciated by one of skill inthis art, any template may be used in the methods and compositions ofthe present invention. Templates which can be mutated and therebyevolved can be used to guide the synthesis of another polypeptide orlibrary of polypeptides as described in the present invention. Asdescribed in more detail herein, the evolvable template encodes thesynthesis of a polypeptide and can be used later to decode the synthetichistory of the polypeptide, to indirectly amplify the polypeptide,and/or to evolve (i.e., diversify, select, and amplify) the polypeptide.The evolvable template is, in certain embodiments, a nucleic acid. Incertain embodiment of the present invention, the template is based on anucleic acid. In other embodiments, the template is a polypeptide.

The nucleic acid templates used in the present invention are made ofDNA, RNA, a hybrid of DNA and RNA, or a derivative of DNA and RNA, andmay be single- or double-stranded. The sequence of the template is usedto encode the synthesis of a polypeptide, preferably a compound that isnot, or does not resemble, a nucleic acid or nucleic acid analog (e.g.,an unnatural polymer or a small molecule). In the case of certainunnatural polymers, the nucleic acid template is used to align themonomer units in the sequence they will appear in the polymer and tobring them in close proximity with adjacent monomer units along thetemplate so that they will react and become joined by a covalent bond.In certain other embodiments, the template can be utilized to generatenon-natural polymers by PCR amplification of a synthetic DNA templatelibrary consisting of a random region of nucleotides.

It will be appreciated that the template can vary greatly in the numberof bases. For example, in certain embodiments, the template may be 10 to10,000 bases long, preferably between 10 and 1,000 bases long. Thelength of the template will of course depend on the length of thecodons, complexity of the library, length of the unnatural polymer to besynthesized, complexity of the small molecule to be synthesized, use ofspace sequences, etc. The nucleic acid sequence may be prepared usingany method known in the art to prepare nucleic acid sequences. Thesemethods include both in vivo and in vitro methods including PCR, plasmidpreparation, endonuclease digestion, solid phase synthesis, in vitrotranscription, strand separation, etc. In certain embodiments, thenucleic acid template is synthesized using an automated DNA synthesizer.

As discussed above, in certain embodiments of the invention, the methodis used to synthesize polypeptides that are not, or do not resemble,nucleic acids or nucleic acid analogs. Thus, in certain embodiments ofthe present invention, the nucleic acid template comprises sequences ofbases that encode the synthesis of an unnatural polymer or smallmolecule. The message encoded in the nucleic acid template preferablybegins with a specific codon that bring into place a chemically reactivesite from which the polymerization can take place, or in the case ofsynthesizing a small molecule the “start” codon may encode for ananti-codon associated with a small molecule scaffold or a firstreactant. The “start” codon of the present invention is analogous to the“start” codon, ATG, which encodes for the amino acid methionine.

In yet other embodiments of the invention, the nucleic acid templateitself may be modified to include an initiation site for polymersynthesis (e.g., a nucleophile) or a small molecule scaffold. In certainembodiments, the nucleic acid template includes a hairpin loop on one ofits ends terminating in a reactive group used to initiate polymerizationof the monomer units. For example, a DNA template may comprise a hairpinloop terminating in a 5′-amino group, which may be protected or not.From the amino group polymerization of the unnatural polymer maycommence. The reactive amino group can also be used to link a smallmolecule scaffold onto the nucleic acid template in order to synthesizea small molecule library.

To terminate the synthesis of the unnatural polymer a “stop” codonshould be included in the nucleic acid template preferably at the end ofthe encoding sequence. The “stop” codon of the present invention isanalogous to the “stop” codons (i.e., TAA, TAG, TGA) found in mRNAtranscripts. These codons lead to the termination of protein synthesis.In certain embodiments, a “stop” codon is chosen that is compatible withthe artificial genetic code used to encode the unnatural polymer. Forexample, the “stop” codon should not conflict with any other codons usedto encode the synthesis, and it should be of the same general format asthe other codons used in the template. The “stop” codon may encode for amonomer unit that terminates polymerization by not providing a reactivegroup for further attachment. For example, a stop monomer unit maycontain a blocked reactive group such as an acetamide rather than aprimary amine. In yet other embodiments, the stop monomer unit comprisesa biotinylated terminus providing a convenient way of terminating thepolymerization step and purifying the resulting polymer.

In one embodiment, mutagenized DNA products are used directly as thetemplate for in vitro synthesis of the corresponding mutant proteins.Because of the high efficiency with which all 19 amino acidsubstitutions can be generated at a single residue, it is possible toperform comprehensive mutagenesis on numerous residues of interest,either independently or in combination with other mutations within theprotein. As used herein, “complete saturation” mutagenesis is defined asreplacing a given amino acid within a protein, with the other 19naturally-occurring amino acids. For example, gene site saturationmutagenesis, which systematically explores minimally all possible singleamino acid substitutions along a protein sequence, is disclosed in Kretzet al., Methods in Enzymology, 2004, 388:3-11; Short, U.S. Pat. No.6,171,820; and Short, U.S. Pat. No. 6,562,594, each of which isincorporated herein by reference. However, these techniques ofsaturation rely upon statistical methods and saturation mutagenesis isnot confirmed by sequencing to assure all intended mutations have beenmade.

In one aspect, this invention provides for the use of codon primers(containing a degenerate N,N,G/T sequence) to introduce point mutationsinto a polynucleotide, so as to generate a set of progeny polypeptidesin which a full range of single amino acid substitutions is representedat each amino acid position (see U.S. Pat. No. 6,171,820; see also, U.S.Pat. No. 5,677,149, each incorporated herein by reference). The oligosused are comprised contiguously of a first homologous sequence, adegenerate N,N,G/T sequence, and preferably but not necessarily a secondhomologous sequence. The downstream progeny translational products fromthe use of such oligos include all possible amino acid changes at eachamino acid site along the polypeptide, because the degeneracy of theN,N,G/T sequence includes codons for all 20 amino acids.

Codon usage is one of the important factors in mammalian geneexpression. The frequencies with which different codons are used varysignificantly between different hosts, and between proteins expressed athigh or low levels within the same organism. The most likely reason forthis variation is that preferred codons correlate with the abundance ofcognate tRNAs available within the cell. It is possible that codon usageand tRNA acceptor concentrations have coevolved, and that the selectionpressure for this co-evolution is more pronounced for highly expressedgenes than genes expressed at low levels.

In one aspect, one such degenerate oligo (comprised of one degenerateN,N,G/T cassette) is used for subjecting each original codon in aparental polynucleotide template to a full range of codon substitutions.In another aspect, at least two degenerate N,N,G/T cassettes areused—either in the same oligo or not, for subjecting at least twooriginal codons in a parental polynucleotide template to a full range ofcodon substitutions. Thus, more than one N,N,G/T sequence can becontained in one oligo to introduce amino acid mutations at more thanone site. This plurality of N,N,G/T sequences can be directlycontiguous, or separated by one or more additional nucleotidesequence(s). In another aspect, oligos serviceable for introducingadditions and deletions can be used either alone or in combination withthe codons containing an N,N,G/T sequence, to introduce any combinationor permutation of amino acid additions, deletions, and/or substitutions.

In another aspect, the present invention provides for the use ofdegenerate cassettes having less degeneracy than the N,N,G/T sequence.For example, it may be desirable in some instances to use (e.g., in anoligo) a degenerate triplet sequence comprised of only one N, where saidN can be in the first second or third position of the triplet. Any otherbases including any combinations and permutations thereof can be used inthe remaining two postitions of the triplet. Alternatively, it may bedesirable in some instances to use (e.g., in an oligo) a degenerateN,N,N triplet sequence.

It is appreciated, however, that the use of a degenerate N,N,G/T tripletas disclosed herein is advantageous for several reasons. In one aspect,this invention provides a means to systematically and fairly easilygenerate the substitution of the full range of possible amino acids (fora total of 20 amino acids) into each and every amino acid position in apolypeptide. Thus, for a 100 amino acid polypeptide, the instantinvention provides a way to systematically and fairly easily generate2000 distinct species (i.e., 20 possible amino acids per position×100amino acid positions). It is appreciated that there is provided, throughthe use of an oligo containing a degenerate N,N,G/T triplet, 32individual sequences that code for 20 possible amino acids. Thus, in areaction vessel in which a parental polynucleotide sequence is subjectedto saturation mutagenesis using one such oligo, there are generated 32distinct progeny polynucleotides encoding 20 distinct polypeptides. Incontrast, the use of a non-degenerate oligo in site-directed mutagenesisleads to only one progeny polypeptide product per reaction vessel.

Thus, in a preferred embodiment, each saturation mutagenesis reactionvessel contains polynucleotides encoding at least 20 progeny polypeptidemolecules such that all 20 amino acids are represented at the onespecific amino acid position corresponding to the codon positionmutagenized in the parental polynucleotide. The 32-fold degenerateprogeny polypeptides generated from each saturation mutagenesis reactionvessel can be subjected to clonal amplification (e.g., cloned into asuitable E. coli host using an expression vector) and subjected toexpression screening. When an individual progeny polypeptide isidentified by screening to display a change in property (when comparedto the template polypeptide), it can be sequenced to identify the aminoacid substitution responsible for such change contained therein.

The template polypeptide may be any protein, however proteins which havea convenient assay for activity such as catalytic activity or ligandbinding are preferred. As used herein, a ligand is any molecule whichbinds specifically to a larger one, such as small molecule binding to aprotein. Representative examples of target interactions includecatalysis, enzyme-substrate interactions, protein-nucleic acidinteractions, receptor-ligand interactions, protein-metal interactionsand antibody-antigen interactions. Representative target proteinsinclude enzymes, antibodies, cytokines, receptors, DNA binding proteins,chelating agents, and hormones.

Any chemical synthetic or recombinant mutagenic method may be used togenerate the population of mutant polypeptides. The practice of thepresent invention may employ, unless otherwise indicated, conventionaltechniques of cell biology, cell culture, molecular biology, transgenicbiology, microbiology, recombinant DNA, and immunology, which are withinthe skill of the art. Such techniques are explained fully in theliterature. See, for example, Molecular Cloning A Laboratory Manual, 2ndEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glovered., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis etal., U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames &S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames &S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, AlanR. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986);B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise,Methods In Enzymology (Academic Press, Inc., N.Y.); Gene TransferVectors For Mammalian Cells (J. H. Miller and M. P. Cabs eds., 1987,Cold Spring Harbor Laboratory); Methods In Enzymnology, Vols. 154 and155 (Wu et al. eds.), Immunochemical Methods In Cell And MolecularBiology (Mayer and Walker, eds., Academic Press, London, 1987); HandbookOf Experimental Immunology, Volumes I-IV (D. M. Weir and C. C.Blackwell, eds., 1986); Manipulating the Mouse Embiyo, (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

In one embodiment, the template polypeptide is an antibody. The antibodyis subjected to the methods described herein to, for example, map andunderstand which positions within the CDR effect binding affinity orwhich positions in the Fc affect expression. The techniques forpreparing and using various antibody-based constructs and fragmentsthereof are well known in the art. An important aspect of the presentinvention is the identification of residues that play, or are likely toplay, a role in the interaction of interest (e.g., antigen-antibodyinteraction, metal chelation, receptor binding, substrate binding, etc).Any antibody or antibody fragment may be used according to the presentinvention.

In one embodiment, any of the evolution platforms CPE, CPI, CPD and CPScan be utilized for generating agonist antibodies, i.e. activatingantibodies. These evolution technologies enable the generation ofagonist antibodies beyond simpler protein crosslinking type activationand in particular allow the activation of receptors such as GPL-1 or 2that are traditionally activated by peptides.

In one aspect, antibodies are selected by FACS or microscopy orequivalent for weakly activating antibodies by using cells withfluorescent signals that fluoresce when the cell surface receptor isactivated. Subsequently, the evolution tools are used to enhance thisactivation. The CPS technology is then utilized to combine up-mutants.

In another aspect, an antibody is selected that binds the receptoractivation site as determined by epitope mapping. CPE, CPI and/or CPDtechniques are used to select for mutants that cause stimulation of thereceptor as determined by an intracellular read-out such as fluorescencein response to calcium ion release or other assays that are well knownin the art. The CPS technology is then utilized to combine up-mutants.

In a particular aspect, some of the key advantages of CPI with single,double or triple amino acid insertions are that these inserted aminoacids can extend into the binding pocket of the receptor to activate thereceptor. In another particular aspect, CPD can remodel and/orreposition amino acids interacting with the receptor to improve oreffect activation and finally CPE can perform relatively smaller changesto effect receptor activation.

The specificity of an antibody is determined by the complementaritydetermining regions (CDRs) within the light chain variable regions (VL)and heavy chain variable regions (VH). The Fab fragment of an antibody,which is about one-third the size of a complete antibody contains theheavy and light chain variable regions, the complete light chainconstant region and a portion of the heavy chain constant region. Fabmolecules are stable and associate well due to the contribution of theconstant region sequences. However, the yield of functional Fabexpressed in bacterial systems is lower than that of the smaller Fvfragment which contains only the variable regions of the heavy and lightchains. The Fv fragment is the smallest portion of an antibody thatstill retains a functional antigen binding site. The Fv fragment has thesame binding properties as the Fab, however without the stabilityconferred by the constant regions, the two chains of the Fv candissociate relatively easily in dilute conditions.

In one aspect, VH and VL regions may be fused via a polypeptide linker(Huston et al., 1991) to stabilize the antigen binding site. This singlepolypeptide Fv fragment is known as a single chain antibody (scFv). TheVH and VL can be arranged with either domain first. The linker joins thecarboxy terminus of the first chain to the amino terminus of the secondchain.

One of skill in the art will recognize that heavy or light chain Fv orFab fragments, or single-chain antibodies may also be used with thissystem. A heavy or light chain can be mutagenized followed by theaddition of the complementary chain to the solution. The two chains arethen allowed to combine and form a functional antibody fragment.Addition of random non-specific light or heavy chain sequences allowsfor the production of a combinatorial system to generate a library ofdiverse members.

Generally, an expression polynucleotide is generated. This expressionpolynucleotide contains: (1) an antibody cassette consisting of a V_(H)domain, spacer peptide, and V_(L) domain operably linked to encode asingle-chain antibody, (2) a promoter suitable for in vitrotranscription (e.g., T7 promoter, SP6 promoter, and the like) operablylinked to ensure in vitro transcription of the single-chain antibodycassette forming a mRNA encoding a single-chain antibody, and (3) atranscription termination sequence suitable for functioning in an invitro transcription reaction. Optionally, the expression polynucleotidemay also comprise an origin of replication and/or a selectable marker.An example of a suitable expression polynucleotide is pLM166.

The V_(H) and V_(L) sequences can be conveniently obtained from alibrary of V_(H) and V_(L) sequences produced by PCR amplification usingV gene family-specific primers or V gene-specific primers (Nicholls etal., J. Immunol. Meth., 1993, 165: 81; WO93/12227) or are designedaccording to standard art-known methods based on available sequenceinformation. Typically, mouse or human V_(H) and V_(L) sequences areisolated. The V_(H) and V_(L) sequences are then ligated, usually withan intervening spacer sequence (e.g., encoding an in-frame flexiblepeptide spacer), forming a cassette encoding a single-chain antibody.Typically, a library comprising a plurality of V_(H) and V_(L) sequencesis used (sometimes also with a plurality of spacer peptide speciesrepresented), wherein the library is constructed with one or more of theV_(H) and V_(L) sequences mutated to increase sequence diversityparticularly at CDR residues, sometimes at framework residues. V regionsequences can be conveniently cloned as cDNAs or PCR amplificationproducts for immunoglobulin-expressing cells. For example, cells fromhuman hybridoma, or lymphoma, or other cell line that synthesizes eithercell surface or secreted immunoglobulin may be used for the isolation ofpolyA+RNA. The RNA is then used for the synthesis of oligo dT primedcDNA using the enzyme reverse transcriptase (for general methods see,Goodspeed et al., Gene 1989, 76: 1; Dunn et al., J. Biol. Chem., 1989,264: 13057). Once the V-region cDNA or PCR product is isolated, it iscloned into a vector to form a single-chain antibody cassette.

To accomplish construction of antibodies and antibody fragments, theencoding genes are isolated and identified. The genes can be modified topermit cloning into an expression vector or an in vitrotranscription/translation. Although methods can be used such as probingthe DNA for VH and VL from hybridoma cDNA (Maniatis et al., 1982) orconstructing a synthetic gene for VH and VL (Barbas et al., 1992), aconvenient mode is to use template directed methods to amplify theantibody sequences. A diverse population of antibody genes can beamplified from a template sample by designing primers to the conservedsequences at the 3′ and 5′ ends of the variable region known as theframework or to the constant regions of the antibody (Iverson et al.,1989). Within the primers, restriction sites can be placed to facilitatecloning into an expression vector. By directing the primers to theseconserved regions, the diversity of the antibody population ismaintained to allow for the construction of diverse libraries. Thespecific species and class of antibody can be defined by the selectionof the primer sequences as illustrated by the large number of sequencesfor all types of antibodies given in Kabat et al., 1987, herebyincorporated by reference.

Messenger RNA isolated from the spleen or peripheral blood of an animalcan be used as the template for the amplification of an antibodylibrary. In certain circumstances, where it is desirable to display ahomogeneous population of antibody fragments on the cell surface, mRNAmay be isolated from a population of monoclonal antibodies. MessengerRNA from either source can be prepared by standard methods and useddirectly or for the preparation of a cDNA template. Generation of mRNAfor cloning antibody purposes is readily accomplished by following thewell-known procedures for preparation and characterization of antibodies(see, e.g., Antibodies: A Laboratory Manual, 1988; incorporated hereinby reference).

Generation of monoclonal antibodies (MAbs) follows generally the sameprocedures as those for preparing polyclonal antibodies. Briefly, apolyclonal antibody is prepared by immunizing an animal with animmunogenic composition in accordance and collecting antisera from thatimmunized animal. A wide range of animal species can be used for theproduction of antisera. Typically the animal used for production ofanti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or agoat. Because of the relatively large blood volume of rabbits, rabbitsare usually preferred for production of polyclonal antibodies.

Immunogenic compositions often vary in immunogenicity. It is oftennecessary therefore to boost the host immune system, as may be achievedby coupling a peptide or polypeptide immunogen to a carrier. Exemplaryand preferred carriers are keyhole limpet hemocyanin (KLH) and bovineserum albumin (BSA). Other albumins such as ovalbumin, mouse serumalbumin or rabbit serum albumin can also be used as carriers. Recognizedmeans for conjugating a polypeptide to a carrier protein are well knownand include glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimideester, carbodiimides and bis-diazotized benzidine.

The immunogenicity of a particular immunogen composition may be enhancedby the use of non-specific stimulators of the immune response, known asadjuvants. Exemplary and preferred adjuvants include complete Freund'sadjuvant (a non-specific stimulator of the immune response containingkilled Mycobacterium tuberculosis), incomplete Freund's adjuvants andaluminum hydroxide adjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster injection, may also be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired level of immunogenicity is obtained, theimmunized animal can be bled and the serum isolated, stored and thespleen harvested for the isolation of mRNA from the polyclonal responseor the animal can be used to generate MAbs for the isolation of mRNAfrom a homogeneous antibody population.

MAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g. a small moleculehapten conjugated to a carrier, a purified or partially purifiedprotein, polypeptide or peptide. The immunizing composition isadministered in a manner effective to stimulate antibody producingcells. Rodents such as mice and rats are frequently used animals;however, the use of rabbit, sheep frog cells is also possible. The useof rats may provide certain advantages (Goding, pp. 60-61, 1986), butmice are preferred, particularly the BALB/c mouse as this is mostroutinely used and generally gives a higher percentage of stablefusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the MAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from blood samples. Spleencells and blood cells are preferable, the former because they are a richsource of antibody-producing cells that are in the dividing plasmablaststage, and the latter because blood is easily accessible. Often, a panelof animals will have been immunized and the spleen of animal with thehighest antibody titer will be removed and the spleen lymphocytesobtained by homogenizing the spleen with a syringe. Typically, a spleenfrom an immunized mouse contains approximately 5×107 to 2×108lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, pp. 65-66, 1986; Campbell, 1984). Forexample, where the immunized animal is a mouse, one may use P3-X63/Ag8,X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG1.7 and S194/5XX0(0 Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3,IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 areall useful in connection with human cell fusions.

One preferred murine myeloma cell is the NS-1 myeloma cell line (alsotermed P3-NS-1-Ag4-1), which is readily available from the NIGMS HumanGenetic Mutant Cell Repository by requesting cell line repository numberGM3573. Another mouse myeloma cell line that may be used is the8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cellline.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 proportion, though the proportion may vary fromabout 20:1 to about 1:1, respectively, in the presence of an agent oragents (chemical or electrical) that promote the fusion of cellmembranes. Fusion methods using Sendai virus have been described byKohler & Milstein (1975; 1976), and those using polyethylene glycol(PEG), such as 37% (v/v) PEG, by Gefter et al., 1977). The use ofelectrically induced fusion methods is also appropriate (Goding pp.71-74, 1986).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. Simple and rapid assaysinclude radioimmunoassays, enzyme immunoassays, cytotoxicity assays,plaque assays, dot immunobinding assays, and the like.

The selected hybridomas are serially diluted and cloned into individualantibody-producing cell lines from which clones can then be propagatedindefinitely to provide MAbs. The cell lines may be exploited for MAbproduction in two basic ways. A sample of the hybridoma can be injected(often into the peritoneal cavity) into a histocompatible animal of thetype that was used to provide the somatic and myeloma cells for theoriginal fusion. The injected animal develops tumors secreting thespecific monoclonal antibody produced by the fused cell hybrid. The bodyfluids of the animal, such as serum or ascites fluid, can then be tappedto provide MAbs in high concentration. The individual cell lines couldalso be cultured in vitro, where the MAbs are naturally secreted intothe culture medium from which they can be readily obtained in highconcentrations. MAbs produced by either means may be further purified,if desired, using filtration, centrifugation and various chromatographicmethods such as HPLC or affinity chromatography.

Following the isolation and characterization of the desired monoclonalantibody, the mRNA can be isolated using techniques well known in theart and used as a template for amplification of the target sequence.

A number of template dependent processes are available to amplify thetarget sequences before and after mutagenesis. One of the best knownamplification methods is the polymerase chain reaction (referred to asPCR) which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202and 4,800,159, and in Innis et al., 1990, each of which is incorporatedherein by reference in its entirety. Briefly, in PCR, two primersequences are prepared which are complementary to regions on oppositecomplementary strands of the target sequence. An excess ofdeoxynucleoside triphosphates are added to a reaction mixture along witha DNA polymerase, e.g., Taq polymerase. If the target sequence ispresent in a sample, the primers will bind to the target and thepolymerase will cause the primers to be extended along the targetsequence by adding on nucleotides. By raising and lowering thetemperature of the reaction mixture, the extended primers willdissociate from the target to form reaction products, excess primerswill bind to the target and to the reaction products and the process isrepeated. Preferably a reverse transcriptase PCR amplification proceduremay be performed in order to quantify the amount of target amplified.Polymerase chain reaction methodologies are well known in the art. Usingenzymatic amplification techniques such as PCR, desired control elementsmay be designed into the primer and thus, will be incorporated into theDNA product.

Another method for amplification is the ligase chain reaction (“LCR”),disclosed in EPA No. 320 308, incorporated herein by reference in itsentirety. In LCR, two complementary probe pairs are prepared, and in thepresence of the target sequence, each pair will bind to oppositecomplementary strands of the target such that they abut. In the presenceof a ligase, the two probe pairs will link to form a single unit. Bytemperature cycling, as in PCR, bound ligated units dissociate from thetarget and then serve as “target sequences” for ligation of excess probepairs. U.S. Pat. No. 4,883,750 describes a method similar to LCR forbinding probe pairs to a target sequence.

Qbeta Replicase, described in PCT Application No. PCT/US87/00880, mayalso be used as an amplification method. In this method, a replicativesequence of RNA which has a region complementary to that of a target isadded to a sample in the presence of an RNA polymerase. The polymerasewill copy the replicative sequence which can then be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[alpha-thio]-triphosphates in one strand of arestriction site may also be useful in the amplification of nucleicacids (Walker et al., 1992).

Strand Displacement Amplification (SDA) is another method of carryingout isothermal amplification of nucleic acids which involves multiplerounds of strand displacement and synthesis, i.e., nick translation. Asimilar method, called Repair Chain Reaction (RCR) involves annealingseveral probes throughout a region targeted for amplification, followedby a repair reaction in which only two of the four bases are present.The other two bases can be added as biotinylated derivatives for easydetection. A similar approach is used in SDA. Target specific sequencescan also be detected using a cyclic probe reaction (CPR). In CPR, aprobe having a 3′ and 5′ sequences of non-specific DNA and middlesequence of specific RNA is hybridized to DNA which is present in asample. Upon hybridization, the reaction is treated with RNaseH, and theproducts of the probe identified as distinctive products which arereleased after digestion. The original template is annealed to anothercycling probe and the reaction is repeated.

Other amplification methods are described in GB Application No. 2 202328, and in PCT Application No. PCT/US89/01025, each of which isincorporated herein by reference in its entirety, may be used inaccordance with the present invention. In the former application,“modified” primers are used in a PCR like, template and enzyme dependentsynthesis. The primers may be modified by labeling with a capture moiety(e.g., biotin) and/or a detector moiety (e.g., enzyme). In the latterapplication, an excess of labeled probes is added to a sample. In thepresence of the target sequence, the probe binds and is cleavedcatalytically. After cleavage, the target sequence is released intact tobe bound by excess probe. Cleavage of the labeled probe signals thepresence of the target sequence.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS), including nucleic acid sequence basedamplification (NASBA) and 3SR (Kwoh et al., 1989). In NASBA, the nucleicacids can be prepared for amplification by standard phenol/chloroformextraction, heat denaturation of a clinical sample, treatment with lysisbuffer and minispin columns for isolation of DNA and RNA or guanidiniumchloride extraction of RNA. These amplification techniques involveannealing a primer which has target specific sequences. Followingpolymerization, DNA/RNA hybrids are digested with RNase H while doublestranded DNA molecules are heat denatured again. In either case thesingle stranded DNA is made fully double-stranded by addition of secondtarget specific primer, followed by polymerization. The double strandedDNA molecules are then multiply transcribed by a polymerase such as T7or SP6. In an isothermal cyclic reaction, the RNAs are reversetranscribed into double stranded DNA, and transcribed once against witha polymerase such as T7 or SP6. The resulting products, whethertruncated or complete, indicate target specific sequences.

Davey et al., EPA No. 329 822 (incorporated herein by reference in itsentirety) disclose a nucleic acid amplification process involvingcyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA, anddouble-stranded DNA (dsDNA), which may be used in accordance with thepresent invention. The ssRNA is a first template for a first primeroligonucleotide, which is elongated by reverse transcriptase(RNA-dependent DNA polymerase). The RNA is then removed from theresulting DNA:RNA duplex by the action of ribonuclease H (RNase H, anRNase specific for RNA in duplex with either DNA or RNA). The resultantssDNA is a second template for a second primer, which also includes thesequences of an RNA polymerase promoter (exemplified by T7 RNApolymerase) 5′ to its homology to the template. This primer is thenextended by DNA polymerase (exemplified by the large “Kienow” fragmentof E. coli DNA polymerase I), resulting as a double-stranded DNA(“dsDNA”) molecule, having a sequence identical to that of the originalRNA between the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle leading to very swift amplification. With properchoice of enzymes, this amplification can be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence can be chosen to be in the form ofeither DNA or RNA.

Miller et al., PCT Application WO 89/06700 (incorporated herein byreference in its entirety) disclose a nucleic acid sequenceamplification scheme based on the hybridization of a promoter/primersequence to a target single-stranded DNA (“ssDNA”) followed bytranscription of many RNA copies of the sequence. This scheme is notcyclic, i.e., new templates are not produced from the resultant RNAtranscripts. Other amplification methods include “race” and “one-sidedPCR” (Frohman, 1990; O'Hara et al., 1989).

Methods based on ligation of two (or more) oligonucleotides in thepresence of nucleic acid having the sequence of the resulting“di-oligonucleotide,” thereby amplifying the di-oligonucleotide, alsomay be used in the amplification step (Wu et al., 1989).

Amplification products may be analyzed by agarose, agarose-acrylamide orpolyacrylamide gel electrophoresis using standard methods (see, e.g.,Maniatis et al., 1982). For example, one may use a 1% agarose gelstained with ethidium bromide and visualized under UV light.Alternatively, the amplification products may be integrally labeled withradio- or fluorometrically-labeled nucleotides. Gels can then be exposedto x-ray film or visualized under the appropriate stimulating spectra,respectively.

Mutagenic procedures of the present invention may comprise any mutagenicapproach that may be tailored to a particular site in a gene, i.e.,site-directed or site-specific mutagenesis. Because the presentinvention relies on comprehensive mutagenesis, the present inventioncontemplates as preferred embodiments those mutagenic procedures thatare rapid, efficient and cost effective.

In one embodiment, the mutagenic procedure utilizes chemical synthesistechniques. In so doing, it is possible to exactly place thesubstitution at one or more particular locations within the gene, andalso to specifically define the nature of the alterations. Chemicalsynthesis methods for DNA are well known within the art. Solid phasetechniques are preferred in this regard.

One advantage to the solid phase method of gene synthesis is theopportunity for mutagenesis using combinatorial synthesis techniques.Combinatorial synthesis techniques are defined as those techniquesproducing large collections or libraries of compounds simultaneously, bysequentially linking different building blocks. Libraries can beconstructed using compounds free in solution, but preferably thecompound is linked to a solid support such as a bead, solid particle oreven displayed on the surface of a microorganism.

Several methods exist for combinatorial synthesis (Holmes et al., 1995;Burbaum et al., 1995; Martin et al., 1995; Freier et al., 1995; Pei etal., 1991; Bruce et al., 1995; Ohlmeyer et al., 1993), including splitsynthesis or parallel synthesis. Split synthesis may be used to producesmall amounts of a relatively large number of compounds, while parallelsynthesis will produce larger amounts of a relatively small number ofcompounds. In general terms, using split synthesis, compounds aresynthesized on the surface of a microparticle. At each step, theparticles are partitioned into several groups for the addition of thenext component. The different groups are then recombined and partitionedto form new groups. The process is repeated until the compound iscompleted. Each particle holds several copies of the same compoundallowing for facile separation and purification. Split synthesis canonly be conducted using a solid support.

An alternative technique known as parallel synthesis may be conductedeither in solid phase or solution. Using parallel synthesis, differentcompounds are synthesized in separate receptacles, often usingautomation. Parallel synthesis may be conducted in microtiter platewhere different reagents can be added to each well in a predefinedmanner to produce a combinatorial library. Parallel synthesis is thepreferred approach for use with enzymatic techniques. It is wellunderstood that many modifications of this technique exist and can beadapted for use with the present invention. Using combinatorial methods,a large number of mutant gene templates may be synthesized.

Mutants genes also may be generated by semisynthetic methods known inthe art (Barbas et al., 1992). Using the conserved regions of anantibody fragment as a framework, variable regions can be inserted inrandom combinations one or more at a time to alter the specificity ofthe antibody fragment and generate novel binding sites, especially inthe generation of antibodies to antigens not conducive to immunizationsuch as toxic or labile compounds. Along the same lines, a knownantibody sequence may be varied by introducing mutations randomly. Thismay be accomplished by methods well known in the art such as the use oferror-prone PCR.

Using the appropriate oligonucleotide primers, PCR is used for the rapidsynthesis of the DNA template containing one or more mutations in thebinding protein gene. Site-specific mutagenesis is a technique useful inthe preparation of individual peptides, or biologically functionalequivalent proteins or peptides, through specific mutagenesis of theunderlying DNA. The technique further provides a ready ability toprepare and test sequence variants, incorporating one or more of theforegoing considerations, by introducing one or more nucleotide sequencechanges into the DNA. Site-specific mutagenesis allows the production ofmutants through the use of specific oligonucleotide sequences whichencode the DNA sequence of the desired mutation, as well as a sufficientnumber of adjacent nucleotides, to provide a primer sequence ofsufficient size and sequence complexity to form a stable duplex on bothsides of the deletion junction being traversed. Typically, a primer ofabout 17 to 25 nucleotides in length is preferred, with about 5 to 10residues on both sides of the junction of the sequence being altered.

The technique typically employs a bacteriophage vector that exists inboth a single stranded and double stranded form. Typical vectors usefulin site-directed mutagenesis include vectors such as the M13 phage.These phage vectors are commercially available and their use isgenerally well known to those skilled in the art. Double strandedplasmids are also routinely employed in site directed mutagenesis, whicheliminates the step of transferring the gene of interest from a phage toa plasmid.

In general, site-directed mutagenesis is performed by first obtaining asingle-stranded vector, or melting of two strands of a double strandedvector which includes within its sequence a DNA sequence encoding thedesired protein. An oligonucleotide primer bearing the desired mutatedsequence is synthetically prepared. This primer is then annealed withthe single-stranded DNA preparation, taking into account the degree ofmismatch when selecting hybridization conditions, and subjected to DNApolymerizing enzymes such as E. coli polymerase I Klenow fragment, inorder to complete the synthesis of the mutation-bearing strand. Thus, aheteroduplex is formed wherein one strand encodes the originalnon-mutated sequence and the second strand bears the desired mutation.This heteroduplex vector is then used to transform appropriate cells,such as E. coli cells, and clones are selected that include recombinantvectors bearing the mutated sequence arrangement.

The preparation of sequence variants of the selected gene usingsite-directed mutagenesis is provided as a means of producingpotentially useful species and is not meant to be limiting, as there areother ways in which sequence variants of genes may be obtained. Forexample, recombinant vectors encoding the desired gene may be treatedwith mutagenic agents, such as hydroxylamine, to obtain sequencevariants.

In certain applications, substitution of amino acids by site-directedmutagenesis, it is appreciated that lower stringency conditions arerequired. Under these conditions, hybridization may occur even thoughthe sequences of probe and target strand are not perfectlycomplementary, but are mismatched at one or more positions. Conditionsmay be rendered less stringent by increasing salt concentration anddecreasing temperature. For example, a medium stringency condition couldbe provided by about 0.1 to 0.25 M NaCl at temperatures of about 37° C.to about 55° C., while a low stringency condition could be provided byabout 0.15 M to about 0.9 M salt, at temperatures ranging from about 20°C. to about 55° C. Thus, hybridization conditions can be readilymanipulated, and thus will generally be a method of choice depending onthe desired results.

In other embodiments, hybridization may be achieved under conditions of,for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 10 mMdithiothreitol, at temperatures between approximately 20° C. to about37° C. Other hybridization conditions utilized could includeapproximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 μM MgCl2, attemperatures ranging from approximately 40° C. to about 72° C. Formamideand SDS also may be used to alter the hybridization conditions.

In a particular embodiment, overlap PCR may be employed. Briefly, aplasmid is used as a template for the first round of PCR. The PCRproducts from the first round are purified and used, together withoutside primers, in the overlap extension PCR reaction. The end productscontained the site directed replacement of a given amino acid with allother possible amino acid residues.

The mutagenized DNA template for the polypeptide of interest can becloned into a plasmid for in vitro transcription/translation or in thepreferred embodiment, the appropriate control elements are includedwithin the PCR product for direct in vitro transcription/translation. Invitro transcription/translation of genes uses cell free extracts toprovide the required enzymes, ribosomes and protein factors. Thesynthesis of proteins is directed by mRNA synthesized from the desiredDNA templates. The DNA template must contain the appropriate controlelements for the system used including a ribosome binding site andpromoter sequence. One of skill in the art would clearly recognize theappropriate required elements for each system.

Prokaryotic in vitro techniques for protein production were the first tobe used (Zubay et al., 1970). Subsequently eukaryotic systems weredeveloped using wheat germ (Roberts, 1973) and rabbit reticulocytes(Pelham, 1976). Several new developments have increased the efficiencyof these techniques. Examples include the development of nucleasedeficient strains of E. coli to improve the results using linear DNAtemplates (Yang, 1980) and treatment of reticulocyte lysates withmicrococcal nuclease to lower any background expression from the system.

The most recent systems developed for in vitro transcription/translationare based on transcription by phage RNA polymerases including SP6 andSP7 (Krieg, 1987, Studier, 1990). DNA placed under the control of T7promoter elements can be used as a template for in vitro transcriptionby T7 RNA polymerase or for complete in vitro transcription/translationwith the polymerase added to either a prokaryotic or eukaryotic proteinsynthesis system. While the methods of the present invention can be usedwith any in vitro transcription/translation system, the T7 system ispreferred for transcription and the use of a prokaryotic translationsystem is preferred as no capping of the RNA is required.

Using in vitro methods for translation, amino acid derivatives may beincorporated into the protein by addition of the derivatized amino acidto the protein synthesis system mixture. Varying the concentration ofthe derivatives, with respect to the normal amino acid, permits one tocreate a mixed population and measure relative effects. G.Characterization

Mutant polypeptides generated by the present invention may becharacterized using a variety of techniques. In general, proteinproducts may be analyzed for the correct apparent molecular weight usingSDS-PAGE. This provides an initial indication that the polypeptide was,in fact, synthesized. When compared to the natural molecule, it alsoindicates whether normal folding or processing is taking place with themutant. In this regard, it may prove useful to label the polypeptide.Alternatively, the polypeptide may be identified by staining of the gel.

Beyond mere synthesis, proteins may be characterized according tovarious properties and an extensive range of functions. Propertiesinclude isoelectric point, thermal stability, sedimentation rate andfolding. One manner of examining folding is the ability to be recognizedby a cognate binding partner. The prime example of this function is theantibody-antigen interaction. A wide variety of different immunoassayformats are available for this purpose and are well known in the art.Principally, changes in either affinity or specificity can be determinedwhen the protein is contacted with a specific ligand or panels ofrelated ligands.

Immunoassays can be generally divided into two types: heterogeneousassays requiring multiple separation steps, and homogeneous assays whichare performed directly. Heterogeneous immunoassays in general involve aligand or antibody immobilized on a solid matrix. A sample containing aligand is contacted with the immobilized antibody and the amount ofcomplex formed on the matrix support is determined from a label attacheddirectly or indirectly to the immobilized complex. As used in thecontext of the present invention, ligand is defined as a species thatinteracts with a non-identical molecule to form a tightly bound, stablecomplex. For practical purposes, the binding affinity is usually greaterthan about 106 M⁻¹ and is preferably in the range of 10⁹-10¹⁵ M⁻¹. Theligand may be any of several types of organic molecules, includingalicyclic hydrocarbons, polynuclear aromatics, halogenated compounds,benzenoids, polynuclear hydrocarbons, nitrogen heterocyclics, sulfurheterocyclics, oxygen heterocyclics, and alkane, alkene alkynehydrocarbons, etc. Biological molecules are of particular interest,including amino acids, peptides, proteins, lipids, saccharides, nucleicacids and combinations thereof. Of course it will be understood thatthese are by way of example only and that contemplated immunoassaymethods are applicable to detecting an extraordinarily wide range ofcompounds, so long as one can obtain an antibody that binds with theligand of interest.

Heterogeneous immunoassays may be performed as sandwich assays in whicha molecule of interest is reacted with an immobilized antibody thatspecifically binds that molecule with high affinity. In a second step, aconjugate formed from the same or different antibody to the antigen anda marker molecule is reacted with the antigen-antibody complex on theimmobilization matrix. After removal of excess free marker conjugate,the bound marker conjugate, which is proportional to the amount ofligand in the sample, is measured.

Detection of immunocomplex formation is well known in the art and may beachieved through the application of numerous approaches. Theseapproaches are typically based upon the detection of a label or marker,such as any of the radioactive, fluorescent, chemiluminescent,electrochemiluminescent, biological or enzymatic tags or labels known inthe art. U.S. patents concerning the use of such labels include U.S.Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;4,275,149 and 4,366,241, each incorporated herein by reference. Ofcourse, one may find additional advantages through the use of asecondary binding ligand such as a second antibody or a biotin/avidinligand binding arrangement, as is known in the art.

Preferred methods for detection includes radioimmunoassay (RIA) orenzyme-linked immunosorbent assay (ELISA) with ELISA being mostpreferred due to generally increased sensitivity. ELISAs are extensivelyused in biotechnology applications, particularly as immunoassays for awide range of antigenic substances. The sensitivity of ELISA is based onthe enzymatic amplification of the signal

Other preferred proteins contemplated for use in accordance with thepresent invention are those which have a convenient assay for activity.Representative examples of target interactions include catalysis,enzyme-substrate interactions, protein-nucleic acid interactions,receptor-ligand interactions and protein-metal interactions. In theseassays the mutant proteins can be compared with the wild-type proteinfor changes in the ability to perform any of the foregoing functions.

As used herein, the term “contacting” is defined as bringing thereaction components into close enough proximity to each other to allowthe desired interaction to occur. Contacting may be accomplished bymixing the components in solution, for example, or by heterogeneousinteraction such as by flow contact through a column or immobilizingmatrix that binds to one of the components.

For mutant proteins having a catalytic activity, the appropriatereaction may be monitored for a change in catalytic rate or analteration in specificity.

The antibodies produced and isolated by the method of the invention areselected to bind a predetermined target. Typically, the predeterminedtarget will be selected in view of its applicability as a diagnosticand/or therapeutic target. The predetermined target may be a known orunknown epitope. Antibodies generally bind to a predetermined antigen(e.g., the immunogen) with an affinity of about at least 1×10⁷ M⁻¹,preferably with an affinity of about at least 5×10⁷ M⁻¹ more preferablywith an affinity of at least 1×10⁸ M-1 to 1×10⁹ M⁻¹ or more, sometimesup to 1×10¹⁰ M⁻¹ or more. Frequently, the predetermined antigen is ahuman protein, such as for example a human cell surface antigen (e.g.,CD4, CD8, IL-2 receptor, EGF receptor, PDGF receptor), other humanbiological macromolecule (e.g., thrombomodulin, protein C, carbohydrateantigen, sialyl Lewis antigen, L-selectin), or nonhuman diseaseassociated macromolecule (e.g., bacterial LPS, virion capsid protein orenvelope glycoprotein) and the like.

In another example, several reports of the diagnostic and therapeuticutility of scFv have been published (Gruber et al., 1994 op.cit.; Lilleyet al., 1994 op.cit.; Huston et al., Int. Rev. Immunol 1993, 10:a 195,Sandhu J S, Crit. Rev. Biotechnol., 1992, 12: 437).

High affinity single-chain antibodies of the desired specificity can beengineered and expressed in a variety of systems. For example, scFv havebeen produced in plants (Firek et al. (1993) Plant Mol. Biol. 23: 861)and can be readily made in prokaryotic systems (Owens R J and Young R J,J. Immunol. Meth., 1994, 168: 149; Johnson S and Bird R E, MethodsEnzymol., 1991, 203: 88). Furthermore, the single-chain antibodies canbe used as a basis for constructing whole antibodies or variousfragments thereof (Kettleborough et al., Euro J. Immunol., 1994, 24:952). The variable region encoding sequence may be isolated (e.g., byPCR amplification or subcloning) and spliced to a sequence encoding adesired human constant region to encode a human sequence antibody moresuitable for human therapeutic uses where immunogenicity is preferablyminimized. The polynucleotide(s) having the resultant fully humanencoding sequence(s) can be expressed in a host cell (e.g., from anexpression vector in a mammalian cell) and purified for pharmaceuticalformulation.

The DNA expression constructs will typically include an expressioncontrol DNA sequence operably linked to the coding sequences, includingnaturally-associated or heterologous promoter regions. Preferably, theexpression control sequences will be eukaryotic promoter systems invectors capable of transforming or transfecting eukaryotic host cells.Once the vector has been incorporated into the appropriate host, thehost is maintained under conditions suitable for high level expressionof the nucleotide sequences, and the collection and purification of themutant “engineered” antibodies.

As stated previously, the DNA sequences will be expressed in hosts afterthe sequences have been operably linked to an expression controlsequence (i.e., positioned to ensure the transcription and translationof the structural gene). These expression vectors are typicallyreplicable in the host organisms either as episomes or as an integralpart of the host chromosomal DNA. Commonly, expression vectors willcontain selection markers, e.g., tetracycline or neomycin, to permitdetection of those cells transformed with the desired DNA sequences(see, e.g., U.S. Pat. No. 4,704,362, which is incorporated herein byreference).

In addition to eukaryotic microorganisms such as yeast, mammalian tissuecell culture may also be used to produce the polypeptides of the presentinvention (see, Winnacker, “From Genes to Clones,” VCH Publishers, N.Y.,N.Y. (1987), which is incorporated herein by reference). Eukaryoticcells are preferred, because a number of suitable host cell linescapable of secreting intact immunoglobulins have been developed in theart, and include the CHO cell lines, various COS cell lines, HeLa cells,myeloma cell lines, transformed B-cells or hybridomas. Expressionvectors for these cells can include expression control sequences, suchas an origin of replication, a promoter, an enhancer (Queen et al.,Immunol. Rev. 1986, 89: 49), and necessary processing information sites,such as ribosome binding sites, RNA splice sites, polyadenylation sites,and transcriptional terminator sequences. Preferred expression controlsequences are promoters derived from immunoglobulin genes,cytomegalovirus, SV40, Adenovirus, Bovine Papilloma Virus, and the like.

Eukaryotic DNA transcription can be increased by inserting an enhancersequence into the vector. Enhancers are cis-acting sequences of between10 to 30 obp that increase transcription by a promoter. Enhancers caneffectively increase transcription when either 5′ or 3′ to thetranscription unit. They are also effective if located within an intronor within the coding sequence itself. Typically, viral enhancers areused, including SV40 enhancers, cytomegalovirus enhancers, polyomaenhancers, and adenovirus enhancers. Enhancer sequences from mammaliansystems are also commonly used, such as the mouse immunoglobulin heavychain enhancer.

Mammalian expression vector systems will also typically include aselectable marker gene. Examples of suitable markers include, thedihydrofolate reductase gene (DHFR), the thymidine kinase gene (TK), orprokaryotic genes conferring drug resistance. The first two marker genesprefer the use of mutant cell lines that lack the ability to growwithout the addition of thymidine to the growth medium. Transformedcells can then be identified by their ability to grow onnon-supplemented media. Examples of prokaryotic drug resistance genesuseful as markers include genes conferring resistance to G418,mycophenolic acid and hygromycin.

The vectors containing the DNA segments of interest can be transferredinto the host cell by well-known methods, depending on the type ofcellular host. For example, calcium chloride transfection is commonlyutilized for prokaryotic cells, whereas calcium phosphate treatment.lipofection, or electroporation may be used for other cellular hosts.Other methods used to transform mammalian cells include the use ofPolybrene, protoplast fusion, liposomes, electroporation, andmicroinjection (see, generally, Sambrook et al., supra).

Once expressed, the antibodies, individual mutated immunoglobulinchains, mutated antibody fragments, and other immunoglobulinpolypeptides of the invention can be purified according to standardprocedures of the art, including ammonium sulfate precipitation,fraction column chromatography, gel electrophoresis and the like (see,generally, Scopes, R., Protein Purification, Springer-Verlag, N.Y.(1982)). Once purified, partially or to homogeneity as desired, thepolypeptides may then be used therapeutically or in developing andperforming assay procedures, immunofluorescent stainings, and the like(see, generally, Immunological Methods, Vols. I and II, Eds. Lefkovitsand Pernis, Academic Press, N.Y. N.Y. (1979 and 1981)).

The oligopeptides of the present invention can be used for diagnosis andtherapy. By way of illustration and not limitation, antibodies can beused to treat cancer, autoimmune diseases, or viral infections. Fortreatment of cancer, the antibodies will typically bind to an antigenexpressed preferentially on cancer cells, such as erbB-2, CEA, CD33, andmany other antigens well known to those skilled in the art. Fortreatment of autoimmune disease, the antibodies will typically bind toan antigen expressed on T-cells, such as CD4, the IL-2 receptor, thevarious T-cell antigen receptors and many other antigens well known tothose skilled in the art (e.g., see Fundamental Immunology, 2nd ed., W.E. Paul, ed., Raven Press: New York, N.Y., which is incorporated hereinby reference). For treatment of viral infections, the antibodies willtypically bind to an antigen expressed on cells infected by a particularvirus such as the various glycoproteins (e.g., gB, gD, gE) of herpessimplex virus and cytomegalovirus, and many other antigens well known tothose skilled in the art (e.g., see Virology, 2nd ed., B. N. Fields etal., eds., (1990), Raven Press: New York, N.Y.).

Pharmaceutical compositions comprising antibodies of the presentinvention are useful for parenteral administration, i.e.,subcutaneously, intramuscularly or intravenously. The compositions forparenteral administration will commonly comprise a solution of theantibody or a cocktail thereof dissolved in an acceptable carrier,preferably an aqueous carrier. A variety of aqueous carriers can beused, e.g., water, buffered water, 0.4% saline, 0.3% glycine and thelike. These solutions are sterile and generally free of particulatematter. These compositions may be sterilized by conventional, well knownsterilization techniques. The compositions may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions such as pH adjusting and buffering agents, toxicity adjustingagents and the like, for example sodium acetate, sodium chloride,potassium chloride, calcium chloride, sodium lactate, etc. Theconcentration of the mutant antibodies in these formulations can varywidely, i.e., from less than about 0.01%, usually at least about 0.1% toas much as 5% by weight and will be selected primarily based on fluidvolumes, viscosities, etc., in accordance with the particular mode ofadministration selected.

Thus, a typical pharmaceutical composition for intramuscular injectioncould be made up to contain 1 ml sterile buffered water, and about 1 mgof mutant antibody. A typical composition for intravenous infusion canbe made up to contain 250 ml of sterile Ringer's solution, and 10 mg ofmutant antibody. Actual methods for preparing parenterally administrablecompositions will be known or apparent to those skilled in the art andare described in more detail in, for example, Remington's PharmaceuticalScience, 20th Ed., Mack Publishing Company, Easton, Pa. (2000), which isincorporated herein by reference.

Biosimilars are protein based therapeutics that have an identical aminoacid sequence (i.e. chemical composition) as an approved ethical drugwhich is no longer patent protected. In one aspect, the techniques ofthe disclosure are utilized for biosimilars. While it is essential toproduce the protein therapeutic in an equivalent formulation andcomposition, to be competitive in the marketplace the biosimilar shouldbe made quickly and as cheaply as possible. Cell culture media andprocess development are some of the most costly and time consuming partsof preparing and producing a biosimilar.

Changing the silent mutation codons within a protein therapeutic changesthe codon used for protein translation but preserve the amino acidsequence within the protein. These codon changes at a variety ofpositions within a molecule, particularly in the amino terminus can havesignificant impact on expression and in some cases even glycosylation.In one aspect, the techniques of the disclosure are utilized in theevolution, selection and preparation of biosimilars.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following examples are to be consideredillustrative and thus are not limiting of the remainder of thedisclosure in any way whatsoever.

EXAMPLES Example 1A Reactions for Comprehensive Positional Insertion(CPI) Evolution, Comprehensive Positional Deletion (CPD) Evolution,Comprehensive Positional Evolution (CPE)

Mutagenesis Reaction

One pair of primers (Primer mix 1 and Primer mix 2) is designed for eachcodon to be mutated. Design will depend on gene sequence, and sequenceanalysis databases such as Sequencher (Gene Codes Corporation) or VectorNTl® (Life Technologies) can be used to design the primers. ForComprehensive Positional Deletion evolution, a degenerate target codon(NNK or NNN) is designed in the middle, flanked by 20 bases on each side(total primer length: 40 bases, 96 clones for sequencing to identifyunique mutants), designed to match the target sequence. For CPE, onepair of primers is designed for each codon to be mutated. A degeneratetarget codon (NNK or NNN) is in the middle, flanked by 20 bases on eachside (total primer length: 43 bases, 9f clones for sequencing toidentify unique mutants). Template DNA is vector DNA with targetgene(s).

Prepare the following reactions in 96-well thin wall PCR plates or 0.2ml thin wall PCR tubes on ice:

Primer mix 1 (2.5 uM) 5 ul Primer mix 2 (2.5 uM) 5 ul 10X Pfu turbo DNApolymerase buffer 2.5 ul DNA template (5, 10, 25 ng) x ul dNTPs 2 ulNuclease-free water QS to 24.5 ul Pfu turbo DNA polymerase (2.5 U/ul)0.5 ul Total reaction volume 25 μl

-   1. Prepare one negative control reaction per one 96-well plate    (replace primers with TE buffer)-   2. Mix gently and spin briefly (5 sec.) in table top centrifuge-   3. Cycle the reactions using the cycling parameters outlined below:

Segment Cycles Temperature Time 1  1 95° C. 30 seconds 2 18 95° C. 30seconds 55° C. 1 minute 68° C. 16 minutes

Quality Control Analysis

-   1. To QC the amplification reactions, set up the following reactions    in 96-well thin wall PCR plates or 0.2 ml thin wall PCR tubes:

Mutagenesis reaction 5 μl Water 4 μl Sample loading buffer 1 μl Volumel0 μl

-   2. Load 10 ul onto a 1% agarose TAE gel with 0.5 μg/ml Ethidium    Bromide. Use 1 kb plus DNA ladder as standard. Run the gel at 100V    for 20-30 minutes in 1×TAE buffer.

Digest the Mutagenesis Reactions with Restriction Enzymes Appropriatefor Cloning into vector DNA—Example for DpnI restriction enzyme

-   1. Add 0.5 μl of the DpnI restriction enzyme (10 U/μl) directly to    each reaction.-   2. Mix gently and spin briefly (5 sec.) in a table top centrifuge-   3. Incubate at 37° C. in PCR machines for 2 hours.-   4. Transform 6 reaction mixtures from each of 96-well plate into XLI    Blue Supercompetent cells. Store the rest of the reactions at −20°    C.-   5. Pre-chill 0.2 ml PCR tubes on ice. Warm SOC medium to 42° C.-   6. Thaw the XLI Blue Supercompetent cells on ice. When thawed,    gently mix and aliquot 50 μl of cells into each of the pre-chilled    tubes.-   7. Add 0.8 ul of b-mercaptoethanol to each aliquot of cells.    Incubate the cells on ice for 10 minutes, swirling gently every 2    minutes.-   8. Add 2 ul of the reaction mixture to one aliquot of cells. Flick    the tubes gently.-   9. Incubate the tubes on cold blocks for 30 minutes.-   10. Heat-pulse the tubes in a 42° C. water bath for 45 seconds.-   11. Incubate the tubes on ice for 2 minutes-   12. Add 100 μl of preheated SOC medium and incubate the tubes at    37° C. for 1 hour with shaking at 225-250 rpm.-   13. Plate the entire transformation mixture on LB agar plates    containing carbenicillin.-   14. Incubate the plates at 37° C. overnight.-   15. Count colonies on plates and pick 12 colonies from each    transformation reaction for miniprep and sequencing.

Large Scale Transformation

-   1. Thaw the XLI Blue Supercompetent cells on ice. Thaw 20 tubes of    competent cells for 96 reactions. When thawed, add 4 μl of    b-mercaptoethanol to each tube of 250 ul competent cells. Incubate    the cells on ice for 10 minutes, swirling gently every 2 minutes.-   2. Pre-chill 0.2 ml PCR tubes on ice. Warm SOC medium to 42° C.-   3. Aliquot 50 μl of cells into each of the pre-chilled tubes.-   4. Add 2 ul of the reaction mixture to one aliquot of cells. Flick    the tubes gently.-   5. Incubate the tubes on cold blocks for 30 minutes.-   6. Heat-pulse the tubes in a 42° C. water bath for 45 seconds.-   7. Incubate the tubes on ice for 2 minutes,-   8. Add 100 μl of preheated SOC medium and incubate the tubes at    37° C. for 1 hour with shaking at 225-250 rpm.-   9. Plate the entire transformation mixture on LB agar plates    containing carbenicillin.-   10. Incubate the plates at 37° C. overnight.-   11. Grow cells for in 96 well blocks for miniprep-   12. Prepare miniprep DNA using QIAVac 96 kit following manufacture's    protocol.

Example 1B Screening for Antibody Affinity Improvement

Transfection

-   -   One week before transfection, transfer 293F cells to monolayer        culture in serum supplemented Dulbecco's Modified Eagle Medium        (D-MEM).    -   One day before transfection, plate 0.2×10⁵ and 0.4×10⁵ cells in        100 ul of serum supplemented D-MEM per transfection sample in 96        well formats.

-   1. For each transfection sample, prepare DNA-Lipofectamine    complexes.

-   2. Dilute 0.2 ug of DNA in 50 ul Opti-MEM Reduced Serum Medium. Mix    gently.

-   3. Dilute 0.125 ul Lipofecctamine in 50 ul Opti-MEM Reduced Serum    Medium. Mix gently and incubate for 5 min at room temperature.

-   4. Combine the diluted DNA with the diluted Lipofectamine. Mix    gently and incubate for 20 min at room temperature.

-   5. Add the 100 ul DNA-Lipofectamine complexes to each well    containing cells and medium.

Mix gently by rocking the plate back and forth.

-   6. Incubate the cells at 37° C. in a 5% CO₂ incubator.-   7. Add 100 vl of serum supplemented D-MEM to each well after 6    hours. Incubate the cells at 37° C. in a 5% CO₂ incubator overnight.-   8. Aspirate off medium in each well. Wash each well with 100 ul of    293 SFM II with 4 mM L-Glutamine. Add 100 ul of 293 SFM II with 4 mM    L-Glutamine to each well.-   9. Collect supernatant for ELISA at 96 hours after transfection.

Functional ELISA

-   1. Coat Nunc-Immuno Maxisorp 96 well plates with 100 ul of 2 ug/ml    antigen in coating solution.-   2. Cover plates with sealers and incubate overnight at 4 C.-   3. Decant plates and tap out residue liquid.-   4. Add 200 uul washing solution. Shake at 200 rpm for 5 min at room    temperature.-   5. Decant plates and tap out residue liquid.-   6. Add 200 ul blocking solution. Shake at 200 rpm for 1 hour at room    temperature.-   7. Decant plates and tap out residue liquid.-   8. Add duplicates of 100 ul/well of control antibody (2 ug/ml) in    blocking solution to the plates.-   9. Add duplicates of 100 ul of supernatant from transfection (SOP    5A) to the plates.-   10. Shake at 200 rpm for one hour at room temperature.-   11. Decant plates and tap out residual liquid.-   12. Add 200 ul washing solution. Shake at 200 rpm for 5 min at room    temperature.-   13. Repeat step 11-12 3 times.-   14. Add 100 ul of 1:5000 dilution of affinity purified goat    anti-human antibody conjugate with HRP in blocking solution to each    well.-   15. Shake at 200 rpm for one hour at room temperature.-   16. Decant plates and tap out residual liquid.-   17. Add 200 ul washing solution. Shake at 200 rpm for 5 min at room    temperature.-   18. Repeat step 17-18 3 times.-   19. Add 100 ul of Sigma TMB substrate to each well. Incubate at room    temperature and check every 2-5 minutes.-   20. Add 100 ul 1N HCl to stop the reaction.-   21. Read at 450 nm.

Quantitation ELISA

-   1. Coat Nunc-Immuno Maxisorp 96 well plates with 100 l of 10 g/ml    affinity-purified Fc-specific goat anti-human IgG in coating    solution.-   2. Cover plates with sealers and incubate overnight at 4 C.-   3. Decant plates and tap out residue liquid.-   4. Add 200 uul washing solution. Shake at 200 rpm for 5 min at room    temperature.-   5. Decant plates and tap out residue liquid.-   6. Add 200 ul blocking solution. Shake at 200 rpm for 1 hour at room    temperature.-   7. Decant plates and tap out residue liquid.-   8. Add duplicates of 100 ul/well of standardized concentration of    purified human serum IgG in blocking solution to the plates.-   9. Add duplicates of 100 ul of supernatant from transfection (SOP    5A) to the plates.-   10. Shake at 200 rpm for one hour at room temperature.-   11. Decant plates and tap out residual liquid.-   12. Add 200 ul washing solution. Shake at 200 rpm for 5 min at room    temperature.-   13. Repeat step 11-12 3 times.-   14. Add 100 ul of 1:5000 dilution of affinity purified goat    anti-human antibody conjugate with HRP in blocking solution to each    well.-   15. Shake at 200 rpm for one hour at room temperature.-   16. Decant plates and tap out residual liquid.-   17. Add 200 ul washing solution. Shake at 200 rpm for 5 min at room    temperature.-   18. Repeat step 17-18 3 times.-   19. Add 100 ul of Sigma TMB substrate to each well. Incubate at room    temperature and check every 2-5 minutes.-   20. Add 100 ul 1N HCl to stop the reaction.-   21. Read at 450 nm

Example 1D Combinatorial Protein Synthesis (CPS)

Combination of Top 10 Single Point Mutants by CPS

In order to further improve the affinity, the top 10 single pointmutants (5 in the light chain, 5 in the heavy chain) can be combined ina combinatorial library, expressed and screened.

The top single point mutants can be combined by a series of PCR/overlapPCR steps as outlined below. Any one of the single point mutants can beused as the template for the initial PCR reactions. In the presentillustration, pBA1 is the template for the initial PCR reactions, andthe single point mutants are in CDRs I, II and III.

All PCR primers are designed to incorporate relevant mutations and matchthe template. Design will depend on gene and sequence analysis databasessuch as Sequencher (Gene Codes Corporation) or Vector NTl® (LifeTechnologies) can be used to design the primers.

1) Combination of Mutants in CDR 1 and CDR3

-   -   a. Perform the 14 PCR reactions for LC and HC: 55° C. annealing,        primers as shown in the table below, template pBA1

Light Chain Light Chain Forward Reverse PCR Forward Reverse PCR PrimerPrimer product primer primer product 1 FP1 L1_R1 299 bp FP2 H1_R1 392 bp2 FP1 L1_R2 299 bp FP2 H1_R2 392 bp 3 FP1 L1_R3 299 bp FP2 H1_R3 392 bp4 FP1 L1_R4 299 bp FP2 H1_R4 392 bp 5 L1_F1 L3_R1 228 bp H1_F1 H3_R1 250bp 6 L1_F2 L3_R1 228 bp H1_F2 H3_R1 250 bp 7 L1_F3 L3_R1 228 bp H1_F3H3_R1 250 bp 8 L1_F4 L3_R1 228 bp H1_F4 H3_R1 250 bp 9 L1_F1 L3_R2 228bp H1_F1 H3_R2 250 bp 10 L1_F2 L3_R2 228 bp H1_F2 H3_R2 250 bp 11 L1_F3L3_R2 228 bp H1_F3 H3_R2 250 bp 12 L1_F4 L3_R2 228 bp H1_F4 H3_R2 250 bp13 L3_F1 RP1 ~1300 bp   H3_F1 RP1 290 bp 14 L3_F2 RP1 ~1300 bp   H3_F2RP1 290 bp

-   -   b. Check PCR reactions on agarose gel    -   c. Pool reactions 1-4, 5-12, and 13-14 for heavy and light        chains in a 1:1 ratio and gel purify full length products (PCR        L1, L2, L3, PCR H1, H2, H3)    -   d. Combine PCR products L1, L2, L3 by overlap extension PCR        using gel purified products from step 1C and primers FP1/RP1    -   e. Combine PCR products H1, H2, H3 by overlap extension PCR        using gel purified products from step 1C and primers FP2/RP1    -   f. Gel purify full length products from steps 1d and 1e        (═Overlap PCR 1; LC: 1.6 kbp, HC: 855 by

2) Addition of Mutants in CDR2

Light Chain Light Chain Forward Reverse PCR Forward Reverse PCR PrimerPrimer product primer primer product 15 FP1 L2_R1   369 bp FP2 H2_R1 487bp 16 FP1 L2_R2   369 bp FP2 H2_R2 487 bp 17 FP1 L2_R3   369 bp FP2H2_R3 487 bp 18 FP1 L2_R4   369 bp FP2 H2_R4 487 bp 19 L2_F1 RP1 ~1450bp H1_F1 RP1 413 bp 20 L2_F2 RP1 ~1450 bp H1_F2 RP1 413 bp 21 L2_F3 RP1~1450 bp H1_F3 RP1 413 bp 22 L2_F4 RP1 ~1450 bp H1_F4 RP1 413 bp

-   -   a. Perform PCR reactions 15-22 for LC and HC: 55° C. annealing,        primers as shown in the table below, template gel purified        overlap PCR product from step if    -   b. Check PCR reaction on agarose gel

c. Pool reactions 15-18, 19-22 for heavy and light chains in a 1:1 ratioand gel purify full length products (PCR L4, L5, PCR H4, H5)

-   -   d. Combine PCR products L4, L5 by overlap extension PCR using        gel purified products from step 2C and primers FP1/RP1    -   e. Combine PCR products H4, H5 by overlap extension PCR using        gel purified products from step 2C and primers FP2/RP1 (LC: 861        bp)    -   f. Gel purify full length products from steps 2d and 2e        (═Overlap PCR 2; LC: 1.6 kbp, HC: 855 bp)        3) Cloning of full length products    -   a. Heavy chains        -   i. Cut HC overlap PCR product from step 2f with RE1/RE2 and            clone into gel purified plasmid cut and CIPed with RE1/RE2        -   ii. Submit 2 96 well plates for sequencing        -   iii. Identify 32 unique HC combinations according to            reference sequences        -   iv. Glycerol stock unique HC combinations and miniprep            plasmid DNA        -   v. Pool HC plasmid DNAs 1:1, cut with RE3/RE4 and gel purify            insert (−2.1 kbp)→pool    -   b. Light chains        -   i. Cut LC overlap PCR product from step 2f with RE5/RE2 and            clone into gel purified plasmid cut and CIPed with RE5/RE2        -   ii. Submit 2 96 well plates to sequencing        -   iii. Identify 32 unique LC combinations according to the            reference sequences        -   iv. Glycerol stock unique LC combinations and miniprep DNAs        -   v. Cut LC DNAs individually with RE3/RE4, CIP and gel purify            vector band

4) Combination of LC and HC

-   -   a. Clone HC pool from step 3a v. into every unique LC from step        3b v.    -   b. Submit 96 clones per LC to sequencing    -   c. Identify unique LC/HC combinations and array in 96 well        plates    -   d. Glycerol stock and miniprep for expression

Example 2 Comprehensive Positional Evolution

This example describes the method of creating specific nucleotidechanges in an antibody construct.

Mutagenesis Reaction

Prepare the following reactions in 96-well thin wall PCR plates or 0.2ml thin wall PCR tubes on ice:

Primer mix 1 (2.5 μM) 5 μl Primer mix 2 (2.5 μM) 5 μl 10X Pfu turbo DNApolymerase buffer 2.5 μl DNA template (5, 10, 25 ng) x μl dNTPs 2 μlNuclease-free water QS to 24.5 μl Pfu turbo DNA polymerase (2.5 U/μl)0.5 μl Total reaction volume 25 μl

-   1. Prepare one negative control reaction per one 96-well plate    (replace primers with TE buffer)-   2. Mix gently and spin briefly (5 sec.) in table top centrifuge-   3. Cycle the reactions using the cycling parameters outlined below:

Segment Cycles Temperature Time 1  1 95° C. 30 seconds 2 18 95° C. 30seconds 55° C. 1 minute 68° C. 16 minutes

Quality Control Analysis

-   1. To QC the amplification reactions, set up the following reactions    in 96-well thin wall PCR plates or 0.2 ml thin wall PCR tubes:

Mutagenesis reaction 5 μl Water 4 μl Sample loading buffer 1 μl Volume10 μl

-   2. Load 10 μl onto a 1% agarose TAE gel with 0.5 μg/ml Ethidium    Bromide. Use 1 kb plus DNA ladder as standard. Run the gel at 100V    for 20-30 minutes in 1×TAE buffer.

Digest the Mutagenesis Reactions with DpnI

-   16. Add 0.5 μl of the DpnI restriction enzyme (10 U/μl) directly to    each reaction.-   17. Mix gently and spin briefly (5 sec.) in a table top centrifuge-   18. Incubate at 37° C. in PCR machines for 2 hours.-   19. Transform 6 reaction mixtures from each of 96-well plate into    XLI Blue Supercompetent cells. Store the rest of the reactions at    −20° C.-   20. Pre-chill 0.2 ml PCR tubes on ice. Warm SOC medium to 42° C.-   21. Thaw the XLI Blue Supercompetent cells on ice. When thawed,    gently mix and aliquot 50 μl of cells into each of the pre-chilled    tubes.-   22. Add 0.8 μl of β-mercaptoethanol to each aliquot of cells.    Incubate the cells on ice for 10 minutes, swirling gently every 2    minutes.-   23. Add 2 μl of the reaction mixture to one aliquot of cells. Flick    the tubes gently.-   24. Incubate the tubes on cold blocks for 30 minutes.-   25. Heat-pulse the tubes in a 42° C. water bath for 45 seconds.-   26. Incubate the tubes on ice for 2 minutes-   27. Add 100 μl of preheated SOC medium and incubate the tubes at    37° C. for 1 hour with shaking at 225-250 rpm.-   28. Plate the entire transformation mixture on LB agar plates    containing carbenicillin.-   29. Incubate the plates at 37° C. overnight.-   30. Count colonies on plates and pick 12 colonies from each    transformation reaction for miniprep and sequencing.

Large Scale Transformation

-   13. Thaw the XLI Blue Supercompetent cells on ice. Thaw 20 tubes of    competent cells for 96 reactions. When thawed, add 4 μl of    β-mercaptoethanol to each tube of 250 ul competent cells. Incubate    the cells on ice for 10 minutes, swirling gently every 2 minutes.-   14. Pre-chill 0.2 ml PCR tubes on ice. Warm SOC medium to 42° C.-   15. Aliquot 50 μl of cells into each of the pre-chilled tubes.-   16. Add 2 μl of the reaction mixture to one aliquot of cells. Flick    the tubes gently.-   17. Incubate the tubes on cold blocks for 30 minutes.-   18. Heat-pulse the tubes in a 42° C. water bath for 45 seconds.-   19. Incubate the tubes on ice for 2 minutes,-   20. Add 100 μl of preheated SOC medium and incubate the tubes at    37° C. for 1 hour with shaking at 225-250 rpm.-   21. Plate the entire transformation mixture on LB agar plates    containing carbenicillin.-   22. Incubate the plates at 37° C. overnight.

Appendix 1 Buffer Recipes

50×TAE Buffer

-   -   242 g Tris base    -   57.1 ml glacial acetic acid    -   37.2 g Na₂EDTA-2H₂O    -   Add distilled H₂O to final volume of 1 liter

1×TAE Buffer

-   -   20 ml 50×TAE buffer    -   800 ml distilled H₂O

1% Agarose Gel with Ethidium Bromide

-   -   1 g LE agarose    -   100 ml 1×TAE buffer    -   Melt the agarose in a microwave oven and swirl to ensure even        mixing    -   Cool agarose to 55° C.    -   Add 2.5 μl of 20 mg/ml Ethidium Bromide to agarose    -   Pour onto a gel platform

LB

-   -   10 g NaCl    -   10 g tryptone    -   5 g yeast extract    -   Add distilled H₂O to a final volume of 1 liter    -   Adjust pH to 7.0 with 5 N NaOH    -   Autoclave

LB-Carbenicillin Agar

-   -   10 g NaCl    -   10 g tryptone    -   5 g yeast extract    -   20 g agar    -   Add distilled H₂O to a final volume of 1 liter    -   Adjust pH to 7.0 with 5 N NaOH    -   Autoclave    -   Cool to 55° C.    -   Add 10 ml of 10 mg/ml of filter-sterilized carbenicillin    -   Pour into petri dishes (25 ml/100-mm plate)

SOC Medium

-   -   0.5 g NaCl    -   20 g tryptone    -   0.5 g yeast extract    -   2 ml of filter-sterilized 20% glucose    -   Add distilled H₂O to a final volume of 1 liter    -   Autoclave    -   Add 10 ml of filter-sterilized 1 M MgCl₂ and 10 ml of        filter-sterilized 1 M MgSO_(s) prior to use

Example 3 Functional ELISA

This example describes the method of comparing the affinity ofantibodies in cell culture supernatant.

-   1. Coat Nunc-Immuno Maxisorp 96 well plates with 100 μl of 2 μg/ml    antigen in coating solution.-   2. Cover plates with sealers and incubate overnight at 4 C.-   3. Decant plates and tap out residue liquid.-   4. Add 200 ul washing solution. Shake at 200 rpm for 5 min at room    temperature.-   5. Decant plates and tap out residue liquid.-   6. Add 200 ul blocking solution. Shake at 200 rpm for 1 hour at room    temperature.-   7. Decant plates and tap out residue liquid.-   8. Add duplicates of 100 ul/well of control antibody (2 μg/ml) in    blocking solution to the plates.-   9. Add duplicates of 100 ul of supernatant from transfection to the    plates.-   10. Shake at 200 rpm for one hour at room temperature.-   11. Decant plates and tap out residual liquid.-   12. Add 200 ul washing solution. Shake at 200 rpm for 5 min at room    temperature.-   13. Repeat step 11-12 3 times.-   14. Add 100 ul of 1:5000 dilution of affinity purified goat    anti-human antibody conjugate with HRP in blocking solution to each    well.-   15. Shake at 200 rpm for one hour at room temperature.-   16. Decant plates and tap out residual liquid.-   17. Add 200 ul washing solution. Shake at 200 rpm for 5 min at room    temperature.-   18. Repeat step 17-18 3 times.-   19. Add 100 ul of Sigma TMB substrate to each well. Incubate at room    temperature and check every 2-5 minutes.-   20. Add 100 ul 1N HCl to stop the reaction.-   21. Read at 450 nm.

Appendix 1 Buffer Recipes

Washing solution

-   -   0.05% Tween-20 in PBS

Blocking Solution

-   -   2% Carnation non-fat milk in PBS

Example 4 CHO-S Cells Transfection

This example describes the method of transfecting DNA into CHO-S cells.

-   1. One week before transfection, transfer CHO-S cells to monolayer    culture in serum supplemented Dulbecco's Modified Eagle Medium    (D-MEM).-   2. One day before transfection, plate 0.4×10⁵ cells in 100 μl of    serum supplemented D-MEM per transfection sample in 96 well formats.-   3. Perform transfection at the end of the work day.-   4. For each transfection sample, prepare DNA-Lipofectamine    complexes.-   5. Dilute 0.2 μg of DNA in 25 μl Opti-MEM Reduced Serum Medium. Mix    gently.-   6. Dilute 0.5 μl Lipofecctamine in 25 μl Opti-MEM Reduced Serum    Medium. Mix gently and incubate for 5 min at room temperature.-   7. Combine the diluted DNA with the diluted Lipofectamine. Mix    gently and incubate for 20 min at room temperature.-   8. Add the 50 μl DNA-Lipofectamine complexes to each well containing    cells and medium. Mix gently by rocking the plate back and forth.-   9. Incubate the cells at 37° C. in a 5% CO₂ incubator overnight-   10. Aspirate off medium in each well. Add 100 μl of serum    supplemented D-MEM to each well.

Collect supernatant for ELISA assay and cell lysate forbeta-galactosidase assay.

Appendix 1 Buffer Recipes

Heat Inactivated Fetal Bovine Serum

-   -   500 ml heat inactivated fetal bovine serum in the original        vendor bottle    -   Heat for 30 minutes at 56° C. with mixing every 5 minutes    -   Prepare 50 ml aliquots and store at −20° C.

Serum Supplemented Dulbecco's Modified Eagle Medium

-   -   500 ml Dulbecco's Modified Eagle Medium    -   50 ml heat inactivated fetal bovine serum    -   5 ml 10 mM MEM Non-Essential Amino Acids

Example 5 Liquid Phase Synthesis of Combinatorial Variable DomainLibraries—Light Chain

This example describes the assembly of a humanized light chain (LC)variable domain library. The library contains human light chainframeworks (FW) and non-human complementarity determining regions (CDR)in the order of: FW1-CDR1-FW2-CDR2-FW3-CDR3. There are total of 7 FW1, 4FW2 and 8 FW3 fragments. The library is assembled by using step wiseliquid phase ligation of FW and CDR DNA fragments.

Assembly of LC Variable Domain

Note 1: Perform Ligation 1 and Ligation 2 at the same time.Note 2: Perform Ligation 3 and Ligation 4 at the same time.

Ligation 1: FW1b→FW1a

-   1. Prepare the following ligation reactions in microcentrifuge tubes    on ice:    -   Note: There are 7 ligation reactions (FW1-1 to FW1-7). Prepare        each ligation reaction in a different microcentrifuge tube,        total of 7 tubes.

FW1a fragments (250 pMole) x μL FW1b fragments (250 pMole) x μL 10X T4ligase Buffer 2 μL 10 mM rATP 1 μL Nuclease-free water QS to 19 μL T4ligase 1 μL Total reaction volume 20 μL

-   2. Mix gently and spin briefly (5 sec.) in microfuge.-   3. Incubate at room temperature for 1 hour.-   4. Set up the following reactions in a 1.5 ml micro-centrifuge tube:

FW1 ligations 20 μl 10x Sample loading buffer 3 μl Total Volume 23 μl

-   5. Load onto a 4% agarose TAE gel with 0.5 μg/ml Ethidium Bromide.    Use 25 bp DNA ladder as standard. Run the gel at 100V for 20-30    minutes in 1×TAE buffer.-   6. Cut out the bands corresponding to the correct sizes and purified    using QIAquick Gel Extraction Kit.-   7. Combine gel fragments from the 7 ligation reactions in two    microcentrifuge tubes.-   8. Add 3 volume of buffer QG to 1 volume of gel.-   9. Incubate at 50° C. for 10 minutes until the gel slice has    completely dissolved. Add 1 gel volume of isopropanol to the sample    and mix.-   10. Place a QIAquick spin column in a provided 2 ml collection tube.-   11. Apply the sample to the QIAquick column, and centrifuge for 1    minute.-   12. Discard flow-through and place QIAquick column back in the same    collection tube.-   13. Add 0.75 ml of buffer PE to QIAquick column and centrifuge for 1    minute.-   14. Discard the flow-through and centrifuge the QIAquick column for    an additional 1 minute at 17,900×g (13,000 rpm).-   15. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.-   16. Add 52 μl of buffer EB to the center of the QIAquick membrane    and centrifuge the column for 1 minute. Let the column stand for 1    minute, and then centrifuge for 1 minute.-   17. Combine the eluted DNA (total volume of 104 μl) and load 6 μl on    4% agarose gel to QC the purified ligation products.

Ligation 2: FW3b→FW3a

-   18. Prepare the following ligation reactions in microcentrifuge    tubes on ice:    -   Note: There are 8 ligation reactions (FW3-1 to FW3-8). Prepare        each ligation reaction in a different microcentrifuge tube,        total of 7 tubes.

FW3a fragments (250 pMole) x μL FW3b fragments (250 pMole) x μL 10X T4ligase Buffer 2 μL 10 mM rATP 1 μL Nuclease-free water QS to 19 μL T4ligase 1 μL Total reaction volume 20 μL

-   19. Mix gently and spin briefly (5 sec.) in microfuge.-   20. Incubate at room temperature for 1 hour.-   21. Set up the following reactions in a 1.5 ml micro-centrifuge    tube:

FW 3 ligations 20 μl 10x Sample loading buffer 3 μl Total Volume 23 μl

-   22. Load onto a 4% agarose TAE gel with 0.5 μg/ml Ethidium Bromide.    Use 25 bp DNA ladder as standard. Run the gel at 100V for 20-30    minutes in 1×TAE buffer.-   23. Cut out the bands corresponding to the correct sizes and    purified using QIAquick Gel Extraction Kit.-   24. Combine gel fragments from the 7 ligation reactions in two    microcentrifuge tubes.-   25. Add 3 volume of buffer QG to 1 volume of gel.-   26. Incubate at 50° C. for 10 minutes until the gel slice has    completely dissolved. Add 1 gel volume of isopropanol to the sample    and mix.-   27. Place a QIAquick spin column in a provided 2 ml collection tube.-   28. Apply the sample to the QIAquick column, and centrifuge for 1    minute.-   29. Discard flow-through and place QIAquick column back in the same    collection tube.-   30. Add 0.75 ml of buffer PE to QIAquick column and centrifuge for 1    minute.-   31. Discard the flow-through and centrifuge the QIAquick column for    an additional 1 minute at 17,900×g (13,000 rpm).-   32. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.-   33. Add 52 μl of buffer EB to the center of the QIAquick membrane    and centrifuge the column for 1 minute. Let the column stand for 1    minute, and then centrifuge for 1 minutes.-   34. Combine the eluted DNA (total volume of 104 μl) and load 6 μl on    4% agarose gel to QC.

Ligation 3: CDR1→FW1

-   1. Prepare ligation reaction in a microcentrifuge tube on ice:

CDR1 fragments (1 nMole) x μL Gel purified combined FW1 fragments 94 μL10X T4 ligase Buffer 14 μL 10 mM rATP 1 μL Nuclease-free water QS to 139μL T4 ligase 1 μL Total reaction volume 140 μL

-   2. Mix gently and spin briefly (5 sec.) in microfuge.-   3. Incubate at room temperature for 1 hour.-   4. Set up the following reactions in a 1.5 ml micro-centrifuge tube:

CDR1-FW 1 ligations 140 μl 10x Sample loading buffer 15 μl Total Volume155 μl

-   5. Load onto a 4% agarose TAE gel with 0.5 μg/ml Ethidium Bromide.    Use 25 bp DNA ladder as standard. Run the gel at 100V for 20-30    minutes in 1×TAE buffer.-   6. Cut out the bands corresponding to the correct sizes and purified    using the QIAquick Gel Extraction Kit.-   7. Combine the gel fragments in two microcentrifuge tubes.-   8. Add 3 volume of buffer QG to 1 volume of gel.-   9. Incubate at 50° C. for 10 minutes until the gel slice has    completely dissolved. Add 1 gel volume of isopropanol to the sample    and mix.-   10. Place a QIAquick spin column in a provided 2 ml collection tube.-   11. Apply the sample to the QIAquick column, and centrifuge for 1    minute.-   12. Discard flow-through and place QIAquick column back in the same    collection tube.-   13. Add 0.75 ml of buffer PE to QIAquick column and centrifuge for 1    minute.-   14. Discard the flow-through and centrifuge the QIAquick column for    an additional 1 minute at 17,900×g (13,000 rpm).-   15. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.-   16. Add 52 μl of buffer EB to the center of the QIAquick membrane    and centrifuge the column for 1 minute. Let the column stand for 1    minute, and then centrifuge for 1 minute.-   17. Combine the eluted DNA (total volume of 104 μl) and load 6 ul on    4% agarose gel to QC.

Ligation 4: CDR2→FW3

-   18. Prepare ligation reaction in a microcentrifuge tube on ice:

CDR2 fragments (1 nMole) x μL Gel purified combined FW3 fragments 94 μL10X T4 ligase Buffer 14 μL 10 mM rATP 1 μL Nuclease-free water QS to 139μL T4 ligase 1 μL Total reaction volume 140 μL

-   19. Mix gently and spin briefly (5 sec.) in microfuge.-   20. Incubate at room temperature for 1 hour.-   21. Set up the following reactions in a 1.5 ml micro-centrifuge    tube:

CDR2-FW3 ligations 140 μl 10x Sample loading buffer 15 μl Total Volume155 μl

-   22. Load onto a 4% agarose TAE gel with 0.5 μg/ml Ethidium Bromide.    Use 25 bp DNA ladder as standard. Run the gel at 100V for 20-30    minutes in 1×TAE buffer.-   23. Cut out the bands corresponding to the correct sizes and    purified using the QIAquick Gel Extraction Kit.-   24. Combine the gel fragments in two microcentrifuge tubes.-   25. Add 3 volume of buffer QG to 1 volume of gel.-   26. Incubate at 50° C. for 10 minutes until the gel slice has    completely dissolved. Add 1 gel volume of isopropanol to the sample    and mix.-   27. Place a QIAquick spin column in a provided 2 ml collection tube.-   28. Apply the sample to the QIAquick column, and centrifuge for 1    minute.-   29. Discard flow-through and place QIAquick column back in the same    collection tube.-   30. Add 0.75 ml of buffer PE to QIAquick column and centrifuge for 1    minute.-   31. Discard the flow-through and centrifuge the QIAquick column for    an additional 1 minute at 17,900×g (13,000 rpm).-   32. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.-   33. Add 52 μl of buffer EB to the center of the QIAquick membrane    and centrifuge the column for 1 minute. Let the column stand for 1    minute, and then centrifuge for 1 minute.-   34. Combine the eluted DNA (total volume of 104 μl) and load 6 μl on    4% agarose gel to QC.

Assembly of LC Variable Domain (Cont.)

Note: Perform Ligation 5 and Ligation 6 at the same time

Ligation 5: FW2→CDR1-FW1

-   1. Prepare ligation reaction in a microcentrifuge tube on ice:

FW2 fragment pool (450 pMole) x μL Gel purified CDR1-FW1 fragments 94 μL10X T4 ligase Buffer 14 μL 10 mM rATP 1 μL Nuclease-free water QS to 139μL T4 ligase 1 μL Total reaction volume 140 μLNote: FW2 fragment pool contain 5 FW2 fragments, each at 90 pMole

-   2. Mix gently and spin briefly (5 sec.) in microfuge.-   3. Incubate at room temperature for 1 hour.-   4. Set up the following reactions in a 1.5 ml micro-centrifuge tube:

FW2-CDR-1-FW1 ligations 140 μl 10x Sample loading buffer 15 μl TotalVolume 155 μl

-   5. Load onto a 4% agarose TAE gel with 0.5 μg/ml Ethidium Bromide.    Use 25 bp DNA ladder as standard. Run the gel at 100V for 20-30    minutes in 1×TAE buffer.-   6. Cut out the bands corresponding to the correct sizes and purified    using QIAquick Gel Extraction Kit.-   7. Combine gel fragments from the 7 ligation reactions in two    microcentrifuge tubes.-   8. Add 3 volume of buffer QG to 1 volume of gel.-   9. Incubate at 50° C. for 10 minutes until the gel slice has    completely dissolved. Add 1 gel volume of isopropanol to the sample    and mix.-   10. Place a QIAquick spin column in a provided 2 ml collection tube.-   11. Apply the sample to the QIAquick column, and centrifuge for 1    minute.-   12. Discard flow-through and place QIAquick column back in the same    collection tube.-   13. Add 0.75 ml of buffer PE to QIAquick column and centrifuge for 1    minute.-   14. Discard the flow-through and centrifuge the QIAquick column for    an additional 1 minute at 17,900×g (13,000 rpm).-   15. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.-   16. Add 30 μl of buffer EB to the center of the QIAquick membrane    and centrifuge the column for 1 minute. Let the column stand for 1    minute, and then centrifuge for 1 minute.-   17. Combine the eluted DNA (total volume of 60 μl) and load 3 μl on    4% agarose gel to QC.

Ligation 6: CDR3→FW3—CDR2

-   18. Prepare ligation reaction in a microcentrifuge tube on ice:

CDR3 fragment pool (500 pMole)  x μL Gel purified FW3-CDR2 fragments 94μL 10X T4 ligase Buffer 14 μL 10 mM rATP  1 μL Nuclease-free water QS to139 μL T4 ligase  1 μL Total reaction volume 140 μL Note: FW2 fragment pool contain 4 FW2 fragments, each at 90 pMole

-   19. Mix gently and spin briefly (5 sec.) in microfuge.-   20. Incubate at room temperature for 1 hour.-   21. Set up the following reactions in a 1.5 ml micro-centrifuge    tube:

CDR3-FW3-CDR2 ligations 140 μl 10x Sample loading buffer  15 μl TotalVolume 155 μl

-   22. Load onto a 4% agarose TAE gel with 0.5 μg/ml Ethidium Bromide.    Use 25 bp DNA ladder as standard. Run the gel at 100V for 20-30    minutes in 1×TAE buffer.-   23. Cut out the bands corresponding to the correct sizes and    purified using QIAquick Gel Extraction Kit.-   24. Combine gel fragments from the 7 ligation reactions in two    microcentrifuge tubes.-   25. Add 3 volume of buffer QG to 1 volume of gel.-   26. Incubate at 50° C. for 10 minutes until the gel slice has    completely dissolved. Add 1 gel volume of isopropanol to the sample    and mix.-   27. Place a QIAquick spin column in a provided 2 ml collection tube.-   28. Apply the sample to the QIAquick column, and centrifuge for 1    minute.-   29. Discard flow-through and place QIAquick column back in the same    collection tube.-   30. Add 0.75 ml of buffer PE to QIAquick column and centrifuge for 1    minute.-   31. Discard the flow-through and centrifuge the QIAquick column for    an additional 1 minute at 17,900×g (13,000 rpm).-   32. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.-   33. Add 30 μl of buffer EB to the center of the QIAquick membrane    and centrifuge the column for 1 minute. Let the column stand for 1    minute, and then centrifuge for 1 minute.-   34. Combine the eluted DNA (total volume of 60 μl) and load 3 μl on    4% agarose gel to QC.

Ligation 7: Full Length LC Variable Domain

-   1. Prepare ligation reactions in a microcentrifuge tube on ice:

FW1-CDR1-FW2 fragments 49 μL CDR2-FW3-CDR3 fragments 49 μL 10X T4 ligaseBuffer 12 μL 10 mM rATP  5 μL Nuclease-free water QS to 345 μL T4 ligase 5 μL Total reaction volume 350 μL 

-   2. Mix gently and spin briefly (5 sec.) in microfuge.-   3. Incubate at room temperature for 1 hour.-   4. Set up the following reactions in a 1.5 ml micro-centrifuge tube:

Full length LC variable domain ligations 140 μl 10x Sample loadingbuffer  15 μl Total Volume 155 μl

-   5. Load onto a 3% agarose TAE gel with 0.5 μg/ml Ethidium Bromide.    Use 100 bp DNA ladder as standard. Run the gel at 100V for 20-30    minutes in 1×TAE buffer.-   6. Cut out the bands corresponding to the correct sizes and purified    using QIAquick Gel

Extraction Kit.

-   7. Combine gel fragments in one microcentrifuge tube.-   8. Add 3 volume of buffer QG to 1 volume of gel.-   9. Incubate at 50° C. for 10 minutes until the gel slice has    completely dissolved. Add 1 gel volume of isopropanol to the sample    and mix.-   10. Place a QIAquick spin column in a provided 2 ml collection tube.-   11. Apply the sample to the QIAquick column, and centrifuge for 1    minute.-   12. Discard flow-through and place QIAquick column back in the same    collection tube.-   13. Add 0.75 ml of buffer PE to QIAquick column and centrifuge for 1    minute.-   14. Discard the flow-through and centrifuge the QIAquick column for    an additional 1 minute at 17,900×g (13,000 rpm).-   15. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.-   16. Add 30 μl of buffer EB to the center of the QIAquick membrane    and centrifuge the column for 1 minute. Let the column stand for 1    minute, and then centrifuge for 1 minute.-   17. Load 3 μl on 3% agarose gel to QC.

Appendix 1 Buffer Recipes

50×TAE Buffer

-   -   242 g Tris base    -   57.1 ml glacial acetic acid    -   37.2 g Na₂EDTA-2H₂O    -   Add distilled H₂O to final volume of 1 liter

1×TAE buffer

-   -   20 ml 50×TAE buffer    -   800 ml distilled H₂O

3% Agarose Gel with Ethidium Bromide

-   -   3 g LE agarose    -   100 ml 1×TAE buffer    -   Melt the agarose in a microwave oven and swirl to ensure even        mixing    -   Cool agarose to 55 C    -   Add 2.5 μl of 20 mg/ml Ethidium Bromide to agarose    -   Pour onto a gel platform

4% Agarose Gel with Ethidium Bromide

-   -   4 g LE agarose    -   100 ml 1×TAE buffer    -   Melt the agarose in a microwave oven and swirl to ensure even        mixing    -   Cool agarose to 55 C    -   Add 2.5 μl of 20 mg/ml Ethidium Bromide to agarose    -   Pour onto a gel platform

Determining the Light Chain CDRs

The following set of rules allows the identification of the CDRs in mostantibody light chain variable domain sequences.

CDR-L1

Start: ˜position 24, always 1 after a cysteine residueResidues before: CLength: 10-17 amino acidsResidues after: always a W, usually W-Y-Q, but also W-L-Q, W-F-Q, W-Y-L

CDR-L2

Start: always 16 residues after the end of CDR-L1Residues before: usually I-Y, but also V-Y, I-K, I-FLength: always 7 amino acids

CDR-L3

Start: always 33 residues after end of CDR-L2 (always 2 after acysteine)Residues before: always CLength: 7-11 residuesResidues after: F-G-X-G (typically F-G-Q-G)

Example 6 β-Galactosidase Assay

This example describes the method for quantitatively measuringβ-galatosidasae expression levels in transfected cells using ONPG as thesubstrate.

-   1. Aspirate the growth medium from the culture dish. Wash 1 time    with 1×PBS-   2. Add 1× Lysis buffer to the culture dish. Use the following    solution volume guideline for various culture dishes:

Type of Volume of 1x culture Lysis Buffer dish (μl/well) 96-well 50plate 24-well 250 plate 12-well 500 plate 6-well plate 1000 60 mm dish2500 100 mm 5000 dish

-   3. Incubate the dish 10-15 minutes at room temperature by swirling    it slowly several times to ensure complete lysis. Observe the    culture dishes under a microscope to confirm that the cells are    lysed completely.    -   Note: Alternatively, freeze the cells for at least one hour at        −20° C. and thaw at room temperature.-   4. Prepare a serial dilution of β-galactosidase standards with    Standard Dilution Buffer separately. A 50 μl aliquot of each point    on the standard curve is transferred to the control wells of the    assay plate. The highest recommended amount of β-galactosidase is    200 milliunits (200,000-400,000 pg). Dilute the standards according    to the guideline below: β-gal Standard Dilution Guide

Standard Dilution β-gal Standard Buffer (miliunits) volume β-galStandard volume 200 990 10 μl of b-gal standard stock 100 200 200 μl of200 mu b-gal standard 50 200 200 μl of 100 mu b-gal standard 25 200 200μl of 50 mu b-gal standard 12.5 200 200 μl of 25 mu b-gal standard 6.25200 200 μl of 12.5 mu b-gal standard 3.125 200 200 μl of 6.25 mu b-galstandard 1.562 200 200 μl of 3.125 mu b-gal standard

-   -   Note 1: Adjust the standard curve to suit the specific        experimental conditions, such as cell types or plasmid vector.    -   Note 2: The dilutions for the standard curve must be prepared        freshly each time the assay is performed.

-   5. Add 50 μl of each sample/well to the assay plate.

-   6. Prepare a blank by adding 50 μl of lysis buffer to a well.

-   7. Add 100 μl of ONPG Substrate Solution to each well. Incubate the    plate at room temperature until the yellow color develops (from    approximately less than one minute to 4 hours depending on the cell    type).

-   8. Read the absorbance at 405-420 nm with a micro-titer    spectrophotometer.

-   9. Quantify β-galactosidase expression based on a linear standard    curve.

Example 7 Antibody Affinity Maturation

This protocol describes the complete process of improving bindingaffinity of an antibody to target antigen.

ds DNA Fragment Preparation

-   1. Order oligonucleotides from IDT (1 μmmol scale, PAGE purified,    lyophilized and 5′ phosphorylated).-   2. Spin down lyophilized oligos in microcentrifuge at 12,000×g for    30 seconds before opening the tubes.-   3. Resuspend oligos in nuclease-free H₂O at 100 pMole/μl according    to the data obtained from IDT-   4. Incubate at 37° C. for 30 min in a thermomixer at 1,000 RPM.-   5. Spin down the re-suspended oligos in microcentrifuge at 12,000×g    for 30 seconds.-   6. Combine 75 μl of matching forward and reverse primers in    thin-wall PCR tubes (or 96 well PCR plates)-   7. Anneal oligonucleotides in a thermocycler using the following    temperature profile: 5′ at 94° C.→5′ at 90° C.→5′ at 85° C.→5′ at    80° C.→5′ at 75° C.→5′ at 70° C.→5′ at 65° C.→5′ at 60° C.→5′ at 55°    C.→5′ at 50° C.→5′ at 45° C.→5′ at 40° C.→5′ at 35° C.→5′ at 30° C.-   8. The final concentration for the annealed DNA fragment    concentration is 50 pMole/μl.-   9. Store the annealed DNA fragments at −20° C.

Quality Control Analysis

-   1. To QC dsDNA fragments (or fragment pools), set up the following    reactions in 1.5 ml micro-centrifuge tubes:

dsDNA fragments  1 μl Water 20 μl Sample loading buffer  1 μl Total 22μl

-   2. Load 10 μl onto a 4% agarose TAE gel with 0.5 μg/ml Ethidium    Bromide. Use 25-bp DNA ladder as standard. Run the gel at 100V for    20-30 minutes in 1×TAE buffer (see Appendix 1).

Appendix 1 Buffer Recipes

50×TAE Buffer

-   -   242 g Tris base    -   57.1 ml glacial acetic acid    -   37.2 g Na₂EDTA-2H₂O    -   Add distilled H₂O to final volume of 1 liter

1×TAE Buffer

-   -   20 ml 50×TAE buffer    -   800 ml distilled H₂O

0.1 M DTT

-   -   1.54 g of DTT    -   10 ml of distilled H₂O    -   Store in −20° C.

80% Glycerol

-   -   20 ml Glycerol    -   80 ml distilled H₂O    -   Sterilize by autoclaving

4% Agarose Gel with Ethidium Bromide

4 g LE agarose

-   -   100 ml 1×TAE buffer    -   Melt the agarose in a microwave oven and swirl to ensure even        mixing    -   Cool agarose to 55 C    -   Add 2.5 μl of 20 mg/ml Ethidium Bromide to agarose    -   Pour onto a gel platform

Example 8 Fully Human Antibody Library Screening

This example describes the method of screening a mammalian cell surfacedisplay fully human antibody library to isolate fully human antibodieswith high specific binding activity to a target antigen using thecombination of flow cytometric sorting and ELISA.

Flow Cytometric Analysis

The screening process need to be optimized for each project according tothe availability of labeled antigens and secondary antibodies. Thisexample was optimized for screening and isolation of high affinityanti-BioAtla 001 fully human antibody.

-   1. Generate fully human antibody libraries stably integrated in    mammalian cells.-   2. Expand stable fully human antibody library clones prior to flow    cytometeric analysis.-   3. On the day of flow cytometric analysis, wash 1×10⁷ cells with    1×PBS-   4. Detach cell with Detachin cell detachment medium and collect    cells in 1×PBS-   5. Spin down cells at 3000 rpm for 5 minutes. Remove supernatant.-   6. Re-suspend cell pellet in 1 ml of cold 1×PBS and spin at 3000 rpm    for 5 minutes.-   7. Remove supernatant and re-suspend the cell pellet in 500 μl of 2    μg/ml of purified human 001 protein in cold 1×PBS.-   8. Incubate on ice for 1 hour with occasionally mixing by hand.-   9. Spin down cells at 3000 rpm for 5 minutes. Remove supernatant.-   10. Re-suspend cell pellet in 1 ml of cold 1×PBS and spin at 3000    rpm for 5 minutes.-   11. Repeat steps 7 and 8.-   12. Remove supernatant and re-suspend the cell pellet in 500 μl of 1    μg/ml of rabbit anti-human 001 polyclonal antibody in cold 1×PBS    with 10% goat serum.-   13. Incubate on ice for 30 minute with occasionally mixing by hand.-   14. Spin down cells at 3000 rpm for 5 minutes. Remove supernatant.-   15. Re-suspend cell pellet in 1 ml of cold 1×PBS and spin at 3000    rpm for 5 minutes.-   16. Repeat steps 7 and 8.-   17. Remove supernatant and re-suspend the cell pellet in 500 μl of    goat anti-rabbit antibody conjugate with FITC and goat anti-human Fc    antibody conjugate with pyroerthrin in cold 1×PBS with 10% goat    serum.-   18. Incubate on ice for 30 minute with occasionally mixing by hand.-   19. Spin down cells at 3000 rpm for 5 minutes. Remove supernatant.-   20. Re-suspend cell pellet in 1 ml of cold 1×PBS and spin at 3000    rpm for 5 minutes.-   21. Repeat steps 7 and 8.-   22. Remove supernatant and re-suspend the cell pellet in 1 ml of    cold 1×PBS with 2% goat serum.-   23. Proceed with flow cytometric analysis using Dako MoFlo.-   24. Draw a sort window to include the top 0.1% of total cells in    terms of ratio of PE/FITC fluorescence. Collect cells that fall    within the sort window in 96 well plates with 100 μl of growth    media.

Recovery of Heavy Chain and Light Chain Variable Region Sequences

-   1. Expand the clones from 96 well plates to 6 well plates. When the    cells reach 80% confluence in the 6 well plates, proceed to genomic    DNA isolation using Qiagen DNeasy Tissue kit.-   2. Aspirate off the media from the cells. Add 500 ml of 1×PBS to    each 6 well. Scrap the cells off the plate with sterile pipet tips.    Transfer scrapped cells in PBS to a sterile micro-centrifuge tube.-   3. Centrifuge the cells for 5 minutes at 3000 rpm.-   4. Remove supernatant and re-suspend cell pellet in 200 μl×PBS.-   5. Add 20 μl proteinase K and 200 μl Buffer AL to the sample, mix    thoroughly by vortexing, and incubate at 56° C. for 10 minutes.-   6. Add 200 μl ethanol to the sample and mix thoroughly by vortexing.-   7. Pipet the mixture from step 6 into a spin column. Centrifuge at    8000 rpm for one minute. Discard the flow-through.-   8. Add 500 μl Buffer AW1 and centrifuge for one minute at 8000 rpm.    Discard the flow-through.-   9. Add 500 μl Buffer AW2 and centrifuge for 2 minutes at 14,000 rpm.    Discard the flow-through. Centrifuge again for one minute at 14,000    rpm. Make sure the membrane is completely dry.-   10. Place the spin column in a sterile micro-centrifuge tube and    pipet 200 μl Buffer AE directly onto the membrane.-   11. Incubate at room temperature for one minute and centrifuge for    one minute at 8000 rpm to elute the genomic DNA.-   12. QC the genomic DNA by setting up the following reactions in 1.5    ml micro-centrifuge tubes:

gDNA  5 μl 10x Sample loading buffer  5 μl Total Volume 10 μlLoad onto a 0.8% agarose TAE gel with 0.5 μg/ml Ethidium Bromide. Use 1kB DNA ladder as standard. Run the gel at 100V for 20-30 minutes in1×TAE buffer.

-   13. Set up the following PCR reactions in sterile PCR tubes:

gDNA   1 μl 2x HotStar Taq Master Mix 12.5 μl Variable domain forwardprimer  0.5 μl Variable domain reverse primer  0.5 μl H2O 10.5 μl TotalVolume   25 μl

-   14. Place the PCR tubes in the thermal cycler and start the cycling    program.    -   Initial activation step: 15 minutes, 95° C.    -   3-step cycling    -   Denaturation: 40 seconds, 94° C.    -   Annealing: 40 seconds, 55° C.    -   Extension: 2 minutes, 72° C.    -   Number of cycles: 30    -   Final extension step: 10 minutes, 72° C.-   15. QC the PCR reactions by setting up the following reactions in    1.5 ml micro-centrifuge tubes:

PCR reaction 5 μl 10x Sample loading buffer 5 μl Total Volume 10 μl 

-   -   Load onto a 1% agarose TAE gel with 0.5 μg/ml Ethidium Bromide.        Use 1 kB DNA ladder as standard. Run the gel at 100V for 20-30        minutes in 1×TAE buffer.

-   16. Set up the following cloning reactions in 1.5 ml    micro-centrifuge tubes using Invitrogen TOPO 2.1 kit:

PCR reaction 4 μl Salt Solution 1 μl TOPO vector 1 μl Total Volume 6 μl

-   17. Mix reactions gently and incubate for 5 minutes at room    temperature.-   18. Add 2 μl of the TOPO cloning reaction from step 17 into a vial    of One Shot Chemically competent E. coli and mix gently.-   19. Incubate on ice for 30 minutes.-   20. Heat-shock the cells for 30 seconds at 42° C.-   21. Transfer the tubes to ice and incubate for 2 minutes.-   22. Add 250 μl of room temperature S.O.C. medium.-   23. Shake the tubes horizontally at 37° C. for one hour at 200 rpm.-   24. Spread 10 μl of the transformation on a re-warmed    LB-carbenicillin plate.-   25. Incubate plate overnight at 37 C.-   26. Pick 6 clones from each transformation for sequencing.-   27. Analyze the heavy chain and light chain variable region    sequences. Proceed to the second round of screening using the ELISA    method.

Digest pBA Vector and Fully Human Antibody Clones with NheI and AgeI

Prepare the following digestion reactions in a microcentrifuge tube onice:

pBAk-LacZ (2 μg) x μl 10X NEB Buffer x 10 μl  Nuclease-free water QS to97 μl Agel (10 U/μl) 3 μl NheI (10 U/μl) 3 μl Total reaction volume 100μl  Fully human antibody clones (5 μg) x μl 10X NEB Buffer x 10 μl Nuclease-free water QS to 97 μl Agel (10 U/μl) 3 μl NheI (10 U/μl) 3 μlTotal reaction volume 100 μl 

-   1. Mix gently and spin briefly (5 sec.) in microfuge-   2. Incubate the reaction at 37° C. overnight

CIP NheI/AgeI Digested pBA Vector and Purify with QIAquick PCRPurification Kit

-   3. Add 2 μl of Apex phosphatase to the microcentrifuge tube    containing the pBAk-LacZ digestion reaction.-   4. Incubate at 37° C. for 10 minutes-   5. Heat at 70° C. for 5 minutes to inactivate the Apex phosphatase-   6. Add 500 μL of Buffer PBI to the microcentrifuge-   7. Mix by vortexing and quick centrifuge-   8. Load 750 μL at a time onto a column-   9. Centrifuge at 12,000×g for 1 minute and decant liquid from    collection tube-   10. Repeat until all sample has been processed.-   11. Wash with 750 μL PE Buffer (Ethanol added!)-   12. Centrifuge at 12,000×g for 1 minute and decant liquid from    collection tube-   13. Place column back onto collection tube and centrifuge again-   14. Put column onto new microcentrifuge tubes and elute with 504, EB    Buffer.

Gel purify NheI/AgeI Digested Fully Human Antibody Clones

-   1. Set up the following reactions in a 1.5 ml micro-centrifuge tube:

NheI/SacII digested Fully human antibody clones 100 μl 10x Sampleloading buffer  3 μl Total Volume 103 μl

-   2. Load onto a 1% agarose TAE gel with 0.5 μg/ml Ethidium Bromide.    Use 1 kB DNA ladder as standard. Run the gel at 100V for 20-30    minutes in 1×TAE buffer.-   3. Cut out the bands corresponding to the heavy chain (HC) and light    chain (LC) variable regions and purified using QIAquick Gel    Extraction Kit.-   4. Add 3 volume of buffer QG to 1 volume of gel.-   5. Incubate at 50° C. for 10 minutes until the gel slice has    completely dissolved. Add 1 gel volume of isopropanol to the sample    and mix.-   6. Place a QIAquick spin column in a provided 2 ml collection tube.-   7. Apply the sample to the QIAquick column, and centrifuge for 1    minute.-   8. Discard flow-through and place QIAquick column back in the same    collection tube.-   9. Add 0.75 ml of buffer PE to QIAquick column and centrifuge for 1    minute.-   10. Discard the flow-through and centrifuge the QIAquick column for    an additional 1 minute at 17,900×g (13,000 rpm).-   11. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.-   12. Add 52 μl of buffer EB to the center of the QIAquick membrane    and centrifuge the column for 1 minute. Let the column stand for 1    minute, and then centrifuge for 1 minute.

Ligate Fully Human HC and LC Variable Domain into NheI/AgeI DigestedpBAk-LacZ Vector

Prepare the following ligation reaction in a microcentrifuge tube onice:

pBAk-LacZ-NheI/AgeI (100 ng) x μl Fully human HC and LC variable domainy μl 5X T4 ligase Buffer 4 μl Nuclease-free water QS to 19 μl T4 ligase(2,000 U/μl) 1 μl Total reaction volume 20 μL

-   1. Mix gently and spin briefly (5 sec.) in microfuge-   2. Incubate at room temperature for 2 hours or 16° C. overnight-   3. Transform each of the ligation reaction mixtures into BioAtla    Supercompetent E. coli cells-   4. Pre-chill 14 ml BD Falcon polypropylene round-bottom tubes on    ice. Prepare SOC medium to 42° C.-   5. Thaw the BioAtla Supercompetent cells on ice. When thawed, gently    mix and aliquot 100ul of cells into each of the pre-chilled tubes.-   6. Add 1.7 μl of β-mercaptoethanol to each aliquot of cells.    Incubate the cells on ice for 10 minutes, swirling gently every 2    minutes.-   7. Add 2 μl of the ligation reaction mixture to one aliquot of    cells. Flick the tubes gently.-   8. Incubate the tubes on ice for 30 minutes.-   9. Heat-pulse the tubes in a 42° C. water bath for 45 seconds.-   10. Incubate the tubes on ice for 2 minutes-   11. Add 900 ul of preheated SOC medium and incubate the tubes at    37° C. for 1 hour with shaking at 225-250 rpm.-   12. Plate 20 μl and 200 μl of the transformation mixture on LB agar    plates containing carbenicillin.-   13. Incubate the plates at 37° C. overnight.-   14. Count colonies on plates and pick 6 colonies for PCR screening    and sequencing.-   15. Choose one clone with the correct sequence, prepare plasmid DNA,    and proceed to transfection in 293F cells.

Transfection of 293F Cells

-   1. One week before transfection, transfer 293F cells to monolayer    culture in serum supplemented Dulbecco's Modified Eagle Medium    (D-MEM).-   2. One day before transfection, plate 0.1×10⁵ cells in 100 μl of    serum supplemented D-MEM per transfection sample in 96 well formats.-   3. For each transfection sample, prepare DNA-Lipofectamine    complexes.-   4. Dilute 0.2 μg of DNA in 50 μl Opti-MEM Reduced Serum Medium. Mix    gently.-   5. Dilute 0.125 μl Lipofecctamine in 50 μl Opti-MEM Reduced Serum    Medium. Mix gently and incubate for 5 min at room temperature.-   6. Combine the diluted DNA with the diluted Lipofectamine. Mix    gently and incubate for 20 min at room temperature.-   7. Add the 100 μl DNA-Lipofectamine complexes to each well    containing cells and medium. Mix gently by rocking the plate back    and forth.-   8. Incubate the cells at 37° C. in a 5% CO₂ incubator.-   9. Add 100 μl of serum supplemented D-MEM to each well after 6    hours. Incubate the cells at 37° C. in a 5% CO₂ incubator overnight.-   10. Aspirate off medium in each well. Wash each well with 100 μl of    293 SFM II with 4 mM L-Glutamine. Add 100 μl of 293 SFM II with 4 mM    L-Glutamine to each well.-   11. Collect supernatant for ELISA at 96 hours after transfection.

Functional ELISA

-   1. Coat Nunc-Immuno Maxisorp 96 well plates with 100 μl of 2 μg/ml    antigen in coating solution.-   2. Cover plates with sealers and incubate overnight at 4 C.

Quantitation ELISA

-   1. Coat Nunc-Immuno Maxisorp 96 well plates with 100 μl of 10 μg/ml    affinity-purified Fc-specific goat anti-human IgG in coating    solution.-   2. Cover plates with sealers and incubate overnight at 4 C.

Functional ELISA

-   3. Decant plates and tap out residue liquid.-   4. Add 200 μl washing solution. Shake at 200 rpm for 5 min at room    temperature.-   5. Decant plates and tap out residue liquid.-   6. Add 200 μl blocking solution. Shake at 200 rpm for 1 hour at room    temperature.-   7. Decant plates and tap out residue liquid.-   8. Add duplicates of 100 μl/well of control antibody (2 μg/ml) in    blocking solution to the plates.-   9. Add duplicates of 100 μl of supernatant from transfection to the    plates.-   10. Shake at 200 rpm for one hour at room temperature.-   11. Decant plates and tap out residual liquid.-   12. Add 200 μl washing solution. Shake at 200 rpm for 5 min at room    temperature.-   13. Repeat step 11-12 3 times.-   14. Add 100 μl of 1:5000 dilution of affinity purified goat    anti-human antibody conjugate with HRP in blocking solution to each    well.-   15. Shake at 200 rpm for one hour at room temperature.-   16. Decant plates and tap out residual liquid.-   17. Add 200 μl washing solution. Shake at 200 rpm for 5 min at room    temperature.-   18. Repeat step 17-18 3 times.-   19. Add 100 μl of Sigma TMB substrate to each well. Incubate at room    temperature and check every 2-5 minutes.-   20. Add 100 μl 1N HCl to stop the reaction.-   21. Read at 450 nm.

Quantitation ELISA

-   1. Decant plates and tap out residue liquid.-   2. Add 200 μl washing solution. Shake at 200 rpm for 5 min at room    temperature.-   3. Decant plates and tap out residue liquid.-   4. Add 200 μl blocking solution. Shake at 200 rpm for 1 hour at room    temperature.-   5. Decant plates and tap out residue liquid.-   6. Add duplicates of 100 μl/well of standardized concentration of    purified human serum IgG in blocking solution to the plates.-   7. Add duplicates of 100 μl of supernatant from transfection to the    plates.-   8. Shake at 200 rpm for one hour at room temperature.-   9. Decant plates and tap out residual liquid.-   10. Add 200 μl washing solution. Shake at 200 rpm for 5 min at room    temperature.-   11. Repeat step 11-12 3 times.-   12. Add 100 μl of 1:5000 dilution of affinity purified goat    anti-human antibody conjugate with HRP in blocking solution to each    well.-   13. Shake at 200 rpm for one hour at room temperature.-   14. Decant plates and tap out residual liquid.-   15. Add 200 μl washing solution. Shake at 200 rpm for 5 min at room    temperature.-   16. Repeat step 17-18 3 times.-   17. Add 100 μl of Sigma TMB substrate to each well. Incubate at room    temperature and check every 2-5 minutes.-   18. Add 100 μl 1N HCl to stop the reaction.-   19. Read at 450 nm.

Appendix 1 Buffer Recipes

1×PBS with 2% Goat Serum

-   -   2 ml goat serum    -   98 ml 1×PBS 50×TAE buffer    -   242 g Tris base    -   57.1 ml glacial acetic acid    -   37.2 g Na₂EDTA-2H₂O    -   Add distilled H₂O to final volume of 1 liter

1×TAE Buffer

-   -   20 ml 50×TAE buffer    -   800 ml distilled H₂O

0.8% Agarose Gel with ethidium bromide

-   -   0.8 g LE agarose    -   100 ml 1×TAE buffer    -   Melt the agarose in a microwave oven and swirl to ensure even        mixing    -   Cool agarose to 55° C.    -   Add 2.5 μl of 20 mg/ml Ethidium Bromide to agarose    -   Pour onto a gel platform

1% Agarose Gel with Ethidium Bromide

-   -   1 g LE agarose    -   100 ml 1×TAE buffer    -   Melt the agarose in a microwave oven and swirl to ensure even        mixing    -   Cool agarose to 55° C.    -   Add 2.5 μl of 20 mg/ml Ethidium Bromide to agarose    -   Pour onto a gel platform

LB

-   -   10 g NaCl    -   10 g tryptone    -   5 g yeast extract    -   Add distilled H₂O to a final volume of 1 liter    -   Adjust pH to 7.0 with 5 N NaOH    -   Autoclave

LB-Carbenicillin Agar

-   -   10 g NaCl    -   10 g tryptone    -   5 g yeast extract    -   20 g agar    -   Add distilled H₂O to a final volume of 1 liter    -   Adjust pH to 7.0 with 5 N NaOH    -   Autoclave    -   Cool to 55° C.    -   Add 10 ml of 10 mg/ml of filter-sterilized carbenicillin    -   Pour into petri dishes (25 ml/100-mm plate)

SOC Medium

-   -   0.5 g NaCl    -   20 g tryptone    -   0.5 g yeast extract    -   2 ml of filter-sterilized 20% glucose    -   Add distilled H₂O to a final volume of 1 liter    -   Autoclave    -   Add 10 ml of filter-sterilized 1 M MgCl₂ and 10 ml of        filter-sterilized 1 M MgSO_(s) prior to use

Washing Solution

-   -   0.05% Tween-20 in PBS

Blocking Solution

-   -   2% Carnation non-fat milk in PBS

Example 9 Synergy Evolution

This example describes the method of creating specific amino acidchanges in a protein expression construct and identifying positions andmutations which do not affect the performance/activity of the targetprotein.

Use CPE to create all 19 single amino acid mutations in the targetmolecule at positions 2-n (n=C-terminal residue of the molecule) or anyother defined range or positions.

Pick 96 clones/codon in deep well plates containing 1200 μA LB withappropriate antibiotic (project/expression construct specific). Sealplates with and grow overnight at 37° C., shaking at 225 RPM.

Replica plate overnight cultures into fresh 96 well plates, growovernight at 37° C.

Miniprep plasmid DNA from overnight cultures (Qiagen endotoxin free96well miniprep kit).

Make glycerol stocks from overnight cultures (replica plates). Transfectclones into HEK293F cells.

Collect supernatant for quant ELISA and project specific functionalELISA.

Appendix 1 Buffer Recipes

50×TAE Buffer

-   -   242 g Tris base    -   57.1 ml glacial acetic acid    -   37.2 g Na₂EDTA-2H₂O    -   Add distilled H₂O to final volume of 1 liter

1×TAE Buffer

-   -   20 ml 50×TAE buffer    -   800 ml distilled H₂O

1% Agarose Gel with Ethidium Bromide

-   -   1 g LE agarose    -   100 ml 1×TAE buffer    -   Melt the agarose in a microwave oven and swirl to ensure even        mixing    -   Cool agarose to 55° C.    -   Add 2.5 μl of 20 mg/ml Ethidium Bromide to agarose    -   Pour onto a gel platform

LB

-   -   10 g NaCl    -   10 g tryptone    -   5 g yeast extract    -   Add distilled H₂O to a final volume of 1 liter    -   Adjust pH to 7.0 with 5 N NaOH    -   Autoclave

LB-Carbenicillin Agar

-   -   10 g NaCl    -   10 g tryptone    -   5 g yeast extract    -   20 g agar    -   Add distilled H₂O to a final volume of 1 liter    -   Adjust pH to 7.0 with 5 N NaOH    -   Autoclave    -   Cool to 55° C.    -   Add 10 ml of 10 mg/ml of filter-sterilized carbenicillin    -   Pour into petri dishes (25 ml/100-mm plate)

SOC Medium

-   -   0.5 g NaCl    -   20 g tryptone    -   0.5 g yeast extract    -   2 ml of filter-sterilized 20% glucose    -   Add distilled H₂O to a final volume of 1 liter    -   Autoclave    -   Add 10 ml of filter-sterilized 1 M MgCl₂ and 10 ml of        filter-sterilized 1 M MgSO_(s) prior to use

Example 10 Generation and Screening of a Fc Codon Variant Library forOptimal Antibody Expression

The present example provides methods for generating a Fc codon variantlibrary and screening methods for obtaining Fc variants with optimizedfor improved expression in production host cells as compare to theparental form of Fc polypeptide.

A. Design and Construction of a Fc Codon Variant Library

For each codon in the target area (in this case the Fc part of the humanIgG1 molecule) a pair of degenerate primers (forward and reverse) isdesigned that includes the target codon and 20 bases on each side. The3^(rd) position of the target codon (wobble position) contains mixedbases (Table 3) that allow the generation of all silent mutations at thetarget position using the same codon (example A). A second set ofdegenerate primer is designed for the same codon position if thecorresponding amino acid can be encoded by another codon (example B).Corresponding forward and reverse degenerate primers are mixed 1:1,annealed to the template and extended to full length products by stranddisplacement using a thermostable DNA polymerase. Template is digestedwith DpnI and full length extension products are transformed into E.coli. Up to 12 colonies per mutagenesis reaction are sequenced. Sequenceconfirmed mutants are arrayed in 96 well plates and glycerol stocked.The glycerol stocks are used to miniprep plasmid DNA for transfectioninto mammalian cells and screening.

TABLE 3 Codes for degenerate bases in synthetic oligos Mixed Symbol BaseR A,G Y C,T M A,C K G,T S C,G W A,T H A,C,T B C,G,T V A,C,G D A,G,T NA,C,G,T Example A: target codon = CCC (proline) → forward primer: CCD,reverse primer: HGG Example B; target codon =TCG (serine) → forwardprimer l: TCH, reverse primer l: DGA → forward primer 2: AGY, reverseprimer 2: RCT 20 bases flanking the target codon are not shown. Totalprimer length: 43 bases.

B. Expression and ELISA Based Screening of Fc Codon Variant Library

Clones from the Fc codon variant library were transfected into amammalian cell line. Full length IgGs were produced and secreted intothe medium. Supernatants of expressed Fc codon variants were screenedfor IgG expression level higher than the parental clone using ELISAassay. The ELISA data was normalized with beta-galactosidase assaymeasuring the transfection efficiency. Top hits identified in theprimary screen were re-transfected and re-screened three times toconfirm the increased expression level. FIG. 3 shows the IgG expressionlevel of the six top hit Fc variants (the six bars on the right) thatare expressed at higher level in a mammalian cell line compare to theparent Fc wild type construct (the bar at left).

1. A method of mapping mutant polypeptides formed from a templatepolypeptide having n amino acid residues, the method comprising: a.generating one of: i. n−1 separate sets of mutant polypeptides, each setcomprising mutant polypeptides having X number of differentpredetermined amino acid residues at a single predetermined position ofthe mutant polypeptide; wherein each set of polypeptides differs in thesingle predetermined position; and the number of different mutantpolypeptides generated is equivalent to (n−1)×X, ii. n−1 or (n−2 in thecase where the initial residue is methionine), separate mutantpolypeptides, wherein each polypeptide differs from the templatepolypeptide in that one amino acid is deleted at a single predeterminedposition, and iii. n+(20×(n−1)) separate mutant polypeptides, whereineach mutant polypeptide differs from the template polypeptide in that ithas inserted after a specific position in the template one each of the20 naturally occurring amino acids; b. assaying each mutant polypeptidefor at least one predetermined property, characteristic or activity; c.for each mutant polypeptide identifying any change in said property,characteristic or activity relative to the template polypeptide; and d.creating a functional map that is used to identify one or more of thegroup consisting of (a) positions and mutations which do not affect theactivity of the mutant polypeptide compared to the template polypeptide;(b) fully mutable sites compared to the template polypeptide; and (c)positions and mutations which result in an up-mutant compared to thetemplate polypeptide. 2-5. (canceled)
 6. The method of claim 1 wherein Xrepresents the 19 naturally occurring amino acid residues not present ina given position of the template polypeptide. 7-12. (canceled)
 13. Themethod of claim 1 wherein said generating step comprises: i. subjectinga codon-containing polynucleotide encoding for said template polypeptideto polymerase-based amplification using a 64-fold degenerateoligonucleotide for each codon to be mutagenized, wherein each of said64-fold degenerate oligonucleotides is comprised of a first homologoussequence and a degenerate N,N,N triplet sequence, so as to generate aset of progeny polynucleotides; and ii. subjecting said set of progenypolynucleotides to clonal amplification such that polypeptides encodedby the progeny polynucleotides are expressed.
 14. The method of claim 1wherein n represents a subset or region of said template polypeptide.15. A method of mapping a set of mutant antibodies formed from atemplate antibody having at least one complementary determining region(CDR), said at least one CDR comprising n amino acid residues, themethod comprising: a. generating n separate sets of mutant antibodies,each set comprising mutant antibodies having X number of differentpredetermined amino acid residues at a single predetermined position ofthe CDR; wherein each set of mutant antibodies differs in the singlepredetermined position; and the number of different mutant antibodiesgenerated is equivalent to n×X; b. assaying each mutant antibody for atleast one predetermined property, characteristic or activity; c. foreach mutant antibody identifying any change in said property,characteristic or activity relative to the template antibody; d.creating a structural positional map; and e. using the structuralpositional map to identify one or more of the group consisting of (a)positions and mutations which do not affect the activity of the mutantantibody compared to the template antibody; (b) fully mutable sitescompared to the template antibody; and (c) positions and mutations whichresult in an up-mutant compared to the template antibody. 16-19.(canceled)
 20. The method of claim 15 wherein said predeterminedproperty, characteristic or activity is at least one of binding affinityand immunogenicity.
 21. The method of claim 15 wherein said templateantibody has six complementary determining regions together comprising namino acid residues 22-25. (canceled)
 26. A functional positional mapmade by the method of claim
 1. 27-28. (canceled)
 29. A method ofproducing a library of multi-site mutant polypeptides formed from atemplate polypeptide having n amino acid residues, the methodcomprising: a. generating n−1 separate sets of point mutationpolypeptides, each set comprising point mutation polypeptides having Xnumber of different predetermined single amino acid substitutions at asingle predetermined site of the polypeptide; wherein each set ofpolypeptides differs in the single predetermined site; and the number ofdifferent point mutation polypeptides generated is equivalent to(n−1)×X; b. screening each point mutation polypeptide for at least onepredetermined property, characteristic or activity; c. identifying anychange in said property, characteristic or activity relative to thetemplate polypeptide for each point mutation polypeptide; d.categorizing each amino acid point mutation as one of deactivating,non-deactivating or activating; e. creating a functional map of suchchanges categorizing each site as one of non-mutable; partially mutable;or fully mutable; f. utilizing the map to select non-deactivating aminoacid point mutations within partially mutable sites that are adjacent tonon-mutable sites; and g. recombining permutations of two to twentyamino acid point mutations selected in step (f) simultaneously togenerate a library of multi-site mutant polypeptides.
 30. The method ofclaim 29 wherein the recombining step utilizes combinatorial proteinsynthesis.
 31. The method of claim 29 wherein contact amino acidresidues in the template polypeptide are categorized as non-mutable inthe functional map.
 32. The method of claim 29 wherein the templatepolypeptide is derived from a portion of an immunoglobulin molecule. 33.The method of claim 32 wherein the portion of the immunoglobulinmolecule is selected from a heavy chain, light chain, variable domain,constant domain, hypervariable region, complementarity determiningregion 1 (CDR1), complementarity determining region 2 (CDR2), andcomplementarity determining region 3 (CDR3).
 34. A method of identifyinga multi-site up-mutant polypeptide, the method comprising: a. generatinga library of multi-site mutant polypeptides; b. screening the library ofmulti-site mutant polypeptides for at least one pre-determined property,characteristic or activity relative to the template polypeptide; and c.identifying the multi-site up-mutant polypeptides as those with one ormore improved properties, characteristics, or activities relative to thetemplate polypeptide.
 35. The method of claim 34 wherein the generatingstep comprises a method of producing a library of multi-site mutantpolypeptides formed from a template polypeptide having n amino acidresidues, the method of producing comprising: a. generating n−1 separatesets of point mutation polypeptides, each set comprising point mutationpolypeptides having X number of different predetermined single aminoacid substitutions at a single predetermined site of the polypeptide;wherein each set of polypeptides differs in the single predeterminedsite; and the number of different point mutation polypeptides generatedis equivalent to (n−1)×X; b. screening each point mutation polypeptidefor at least one predetermined property, characteristic or activity; c.identifying any change in said property, characteristic or activityrelative to the template polypeptide for each point mutationpolypeptide; d. categorizing each amino acid point mutation as one ofdeactivating, non-deactivating or activating; e. creating a functionalmap of such changes categorizing each site as one of non-mutable;partially mutable; or fully mutable; f. utilizing the map to selectnon-deactivating amino acid point mutations within partially mutablesites that are adjacent to non-mutable sites; and g. recombiningpermutations of two to twenty amino acid point mutations selected instep (f) simultaneously to generate a library of multi-site mutantpolypeptides.
 36. A method of selecting two or more sites in a templatepolypeptide for multi-site mutation, the method comprising: a. obtaininga functional map of mutant polypeptides formed from a templatepolypeptide; b. utilizing the map to select partially mutable sites thatare adjacent to non-mutable sites for multi-site mutation.
 37. Themethod of claim 36 wherein the obtaining step comprises a method ofmapping mutant polypeptides formed from a template polypeptide having namino acid residues, the method of mapping comprising: a. generating n−1separate sets of mutant polypeptides, each set comprising mutantpolypeptides having X number of different predetermined amino acidresidues at a single predetermined position of the polypeptide; whereineach set of mutant polypeptides differs in the single predeterminedposition; and the number of different member polypeptides generated isequivalent to (n−1)×X; b. assaying each mutant polypeptide for at leastone predetermined property, characteristic or activity; c. for eachmutant polypeptide identifying any change in said property,characteristic or activity relative to the template polypeptide; and d.creating a functional map of such changes identifying positions andmutations which do not affect the activity of the mutant polypeptidecompared to the template polypeptide. 38-50. (canceled)
 51. A method ofproviding an optimized protein, the method comprising: a. selecting atemplate polypeptide having n amino acid residues; b. generating n−1separate sets of mutant polypeptides from the template polypeptide, eachset comprising mutant polypeptides having X number of differentpredetermined amino acid residues at a single predetermined position ofthe polypeptide; wherein each set of polypeptides differs in the singlepredetermined position; and the number of different mutant polypeptidesgenerated is equivalent to (n−1)×X; c. assaying each mutant polypeptidefor at least one predetermined property, characteristic or activity; d.identifying any change in said property, characteristic or activity ofthe mutant polypeptide relative to the template polypeptide; e. creatinga functional map wherein the functional map is used to identifypositions and mutations which result in an up-mutant compared to thetemplate polypeptide; f. combining two or more of each of the relevantpositions and mutations identified from mutant polypeptides which resultin a up-mutant compared to the template polypeptide to create a set ofcombinatorial polypeptides; g. assaying each combinatorial polypeptidefor at least one predetermined property, characteristic or activity; andh. selecting a combinatorial polypeptide in which the predeterminedproperty, characteristic or activity is optimized compared to up-mutantmutant polypeptides.
 52. The method of claim 1, wherein the generatingstep (a) further comprises confirming by sequencing, or some othermethod, the presence of the intended predetermined amino acid residue atthe single predetermined position in each mutant polypeptide in each setof mutant polypeptides.
 53. The method of claim 15, wherein thegenerating step (a) further comprises confirming by sequencing, or someother method, the presence of the intended predetermined amino acidresidue at the single predetermined position of the CDR in each mutantantibody in each set of mutant antibodies.
 54. The method of claim 29wherein the generating step (a) further comprises confirming bysequencing, or some other method, the presence of each of the intendedpredetermined single amino acid substitutions at the predetermined sitein each point mutation polypeptide in each set of point mutationpolypeptides.
 55. The method of claim 34 wherein the generating step (a)further comprises confirming the sequence of each multi-site mutantpolypeptide in the library by sequencing, or some other method. 56.(canceled)
 57. The method of claim 41 wherein the generating step (a)further comprises confirming by sequencing, or some other method, theintended amino acid deletion at the single predetermined position. 58.The method of claim 46 wherein the generating step (a) further comprisesconfirming by sequencing, or some other method, the intended amino acidinsertion at the specific position in the template.
 59. A method ofevolving a polypeptide encoding a functional protein, the methodcomprising: a. generating and cloning at least 20 or more separate setsof mutant polypeptides, each set comprising mutant polypeptides havingfrom 5 to 19 different predetermined amino acid residues at a singlepredetermined position of the polypeptide; wherein each set of mutantpolypeptides differs in the single predetermined position; b. confirmingthat each different mutant polypeptide was generated and cloned; and c.assaying each set for at least one predetermined property,characteristic or activity.
 60. The method of claim 59, wherein in step(a), each set of mutant polypeptides comprises mutant polypeptideshaving from 10 to 19, from 15 to 19, or 19 different predetermined aminoacid residues at a single predetermined position of the polypeptide. 61.The method of claim 59, further comprising: d. performing bioinformaticanalysis to identify positions of potential interest for furthermodification; and e. mutating positions identified in step d.
 62. Themethod of claim 61 wherein bioinformatic analysis is generation of afunctional map.
 63. The method of claim 59 wherein the at least onepredetermined property, characteristic or activity is modifiedexpression
 64. The method of claim 63 wherein the modified expression isimproved expression.
 65. A method of evolving a polypeptide encoding afunctional protein and having n amino acid residues, the methodcomprising: a. generating and cloning n−1 sets of mutant polypeptides,each set comprising mutant polypeptides having 19 differentpredetermined amino acid residues at a single predetermined position ofthe polypeptide; wherein each set of mutant polypeptides differs in thesingle predetermined position; and the total number of different mutantpolypeptides generated is equivalent to (n−1)×19; b. confirming thateach different mutant polypeptide was generated and cloned; and c.assaying each set for at least one predetermined property,characteristic or activity;
 66. The method of claim 65, the methodfurther comprising: d. performing bioinformatic analysis to identifypositions of potential interest for further modification; and e.mutating positions identified in step d.
 67. The method of claim 66wherein the bioinformatic analysis is generation of a functional map.68. The method of claim 65 wherein the at least one predeterminedproperty, characteristic or activity is modified expression.
 69. Themethod of claim 68 wherein the modified expression is improvedexpression.
 70. A method of evolving a polynucleotide encoding afunctional protein, the method comprising: a. incorporating N,N,N at apredetermined codon position in the polynucleotide in order to generateone or more predetermined codon mutants within a set of predeterminedmutant polynucleotides and cloning each predetermined mutantpolynucleotide; b. confirming that each predetermined mutantpolynucleotide in the set was generated and cloned; c. producing a setof predetermined polypeptides from set of predetermined polynucleotides;and d. assaying each predetermined polypeptide for at least onepredetermined property, characteristic or activity compared to thefunctional protein.
 71. The method of claim 70 wherein the at least onepredetermined property, characteristic or activity is modifiedexpression.
 72. The method of claim 71 wherein the modified expressionis improved expression.
 73. The method of claim 70 wherein theconfirming step comprises sequencing of each mutant polynucleotide. 74.A method of evolving a polypeptide from a functional protein, the methodcomprising: a. incorporating N,N,N at every codon position in apolynucleotide encoding the functional protein in order to generate aset of predetermined mutant polynucleotides containing every possiblecodon sequence and cloning each predetermined mutant polynucleotide; b.confirming by sequencing, or some other method, that greater than 75%,50%, 25%, or 1% of the predetermined mutant polynucleotides in the setwas generated and cloned; c. producing a set of predetermined mutantpolypeptides from the set of predetermined mutant polynucleotides; andd. assaying each predetermined mutant polypeptide for at least onepredetermined property, characteristic or activity compared to thefunctional protein. 75-76. (canceled)
 77. A functional positional mapmade by the method of claim
 15. 78. The method of claim 37, wherein thegenerating step (a) further comprises confirming by sequencing, or someother method, the presence of the intended predetermined amino acidresidue at the single predetermined position in each mutant polypeptidein each set of mutant polypeptides.
 79. The method of claim 27, whereinthe generating step (a) further comprises confirming by sequencing, orsome other method, the presence of the intended predetermined amino acidresidue at the single predetermined position of the CDR in each mutantantibody in each set of mutant antibodies.
 80. The method of claim 1,wherein in step (a), n−1 separate sets of mutant polypeptides, each setcomprising mutant polypeptides having X number of differentpredetermined amino acid residues at a single predetermined position ofthe mutant polypeptide; wherein each set of polypeptides differs in thesingle predetermined position; and the number of different mutantpolypeptides generated is equivalent to (n−1)×X, are generated.
 81. Themethod of claim 1, wherein in step (a), n−1 or (n−2 in the case wherethe initial residue is methionine), separate mutant polypeptides,wherein each polypeptide differs from the template polypeptide in thatone amino acid is deleted at a single predetermined position, aregenerated.
 82. The method of claim 1, wherein in step (a), n+(20×(n−1))separate mutant polypeptides, wherein each mutant polypeptide differsfrom the template polypeptide in that it has inserted after a specificposition in the template one each of the 20 naturally occurring aminoacids, are generated.